Prevention of primary and metastatic neoplastic diseases with hsp90-peptide complexes

ABSTRACT

The present invention relates to methods and compositions for eliciting an immune response and the prevention and treatment of primary and metastatic neoplastic diseases and infectious diseases. The methods of the invention comprise administering a composition comprising an effective amount of a complex, in which the complex consists essentially of a heat shock protein (hsp) noncovalently bound to an antigenic molecule. Optionally, the methods further comprise administering antigen presenting cells sensitized with complexes of hsps noncovalently bound to an antigenic molecule. “Antigenic molecule” as used herein refers to the peptides with which the hsps are endogenously associated in vivo as well as exogenous antigens/immunogens (i.e., with which the hsps are not complexed in vivo) or antigenic/immunogenic fragments and derivatives thereof. In a preferred embodiment, the complex is autologous to the individual. In a specific embodiment; the effective amounts of the complex are in the range of 0.1 to 9.0 micrograms for complexes comprising hsp70, 5 to 49 micrograms for hsp90, and 0.1 to 9.0 micrograms for gp96.

This is a division of application No. 08/796,319, filed Feb. 7, 1997 nowU.S. Pat. No. 6,017,540, which is incorporated by reference herein inits entirety.

This invention was made with government support under grant numbersCA44786 and CA64394 awarded by the National Institutes of Health. Thegovernment has certain rights in the invention.

1. INTRODUCTION

The present invention relates to methods and compositions for theprevention and treatment of infectious diseases, primary and metastaticneoplastic diseases, including, but not limited to human sarcomas andcarcinomas. In the practice of the prevention and treatment ofinfectious diseases and cancer, compositions of complexes of heatshock/stress proteins (hsps) including, but not limited to, hsp70,hsp90, gp96 alone or in combination with each other, noncovalently boundto antigenic molecules, are used to augment the immune response togenotoxic and nongenotoxic factors, tumors and infectious agents. In thepractice of the invention, hsp-antigenic molecule complexes may beadministered alone or in combination with the administration of antigenpresenting cells sensitized with an hsp-antigenic molecule complex.

2. BACKGROUND OF THE INVENTION

The era of tumor immunology began with experiments by Prehn and Main,who showed that antigens on the methylcholanthrene (MCA)-inducedsarcomas were tumor specific in that transplantation assays could notdetect these antigens in normal tissue of the mice (Prehn, R. T., etal., 1957, J. Natl. Cancer Inst. 18:769-778). This notion was confirmedby further experiments demonstrating that tumor specific resistanceagainst MCA-induced tumors can be elicited in the autochthonous host,that is, the mouse in which the tumor originated (Klein, G., et al.,1960, Cancer Res. 20:1561-1572).

In subsequent studies, tumor specific antigens were also found on tumorsinduced with other chemical or physical carcinogens or on spontaneoustumors (Kripke, M. L., 1974, J. Natl. Cancer Inst. 53:1333-1336; Vaage,J., 1968, Cancer Res. 28:2477-2483; Carswell, E. A., et al., 1970, J.Natl. Cancer Inst. 44:1281-1288). Since these studies used protectiveimmunity against the growth of transplanted tumors as the criterion fortumor specific antigens, these antigens are also commonly referred to as“tumor specific transplantation antigens” or “tumor specific rejectionantigens.” Several factors can greatly influence the immunogenicity ofthe tumor induced, including, for example, the specific type ofcarcinogen involved, immunocompetence of the host and latency period(Old, L. J., et al., 1962, Ann. N. Y. Acad. Sci. 101:80-106; Bartlett,G. L., 1972, J. Natl. Cancer Inst. 49:493-504).

Most, if not all, carcinogens are mutagens which may cause mutation,leading to the expression of tumor specific antigens (Ames, B. N., 1979,Science 204:587-593; Weisburger, J. H., et al., 1981, Science214:401-407). Some carcinogens are immunosuppressive (Malmgren, R. A.,et al., 1952, Proc. Soc. Exp. Biol. Med. 79:484-488). Experimentalevidence suggests that there is a constant inverse correlation betweenimmunogenicity of a tumor and latency period (time between exposure tocarcinogen and tumor appearance) (Old, L. J., et al., 1962, Ann. N.Y.Acad. Sci. 101:80-106; and Bartlett, G. L., 1972, J. Natl. Cancer Inst.49:493-504). Other studies have revealed the existence of tumor specificantigens that do not lead to rejection, but, nevertheless, canpotentially stimulate specific immune responses (Roitt, I., Brostoff, Jand Male, D., 1993, Immunology, 3rd ed., Mosby, St. Louis, pps.17.1-17.12).

2.1. Tumor-Specific Immunogenicities of Heat Shock/Stress Proteinshsp70, hsp90 and gp96

Srivastava et al. demonstrated immune response tomethylcholanthrene-induced sarcomas of inbred mice (1988, Immunol. Today9:78-83). In these studies it was found that the molecules responsiblefor the individually distinct immunogenicity of these tumors wereidentified as cell-surface glycoproteins of 96 kDa (gp96) andintracellular proteins of 84 to 86 kDa (Srivastava, P. K., et al., 1986,Proc. Natl. Acad. Sci. USA 83:3407-3411; Ullrich, S. J., et al., 1986,Proc. Natl. Acad. Sci. USA 83:3121-3125. Immunization of mice with gp96or p84/86 isolated from a particular tumor rendered the mice immune tothat particular tumor, but not to antigenically distinct tumors.Isolation and characterization of genes encoding gp96 and p84/86revealed significant homology between them, and showed that gp96 andp84/86 were, respectively, the endoplasmic reticular and cytosoliccounterparts of the same heat shock proteins (Srivastava, P. K., et al.,1988, Immunogenetics 28:205-207; Srivastava, P. K., et al., 1991, Curr.Top. Microbiol. Immunol. 167:109-123). Further, hsp70 was shown toelicit immunity to the tumor from which it was isolated but not toantigenically distinct tumors. However, hsp70 depleted of peptides wasfound to lose its immunogenic activity (Udono, M., and Srivastava, P.K., 1993, J. Exp. Med. 178:1391-1396). These observations suggested thatthe heat shock proteins are not immunogenic per se, but are carriers ofantigenic peptides that elicit specific immunity to cancers (Srivastava,P. K., 1993, Adv. Cancer Res. 62:153-177).

2.2. Pathobiolopy of Cancer

Cancer is characterized primarily by an increase in the number ofabnormal cells derived from a given normal tissue, invasion of adjacenttissues by these abnormal cells, and lymphatic or blood-borne spread ofmalignant cells to regional lymph nodes and to distant sites(metastasis). Clinical data and molecular biologic studies indicate thatcancer is a multistep process that begins with minor preneoplasticchanges, which may under certain conditions progress to neoplasia.

Pre-malignant abnormal cell growth is exemplified by hyperplasia,metaplasia, or most particularly, dysplasia (for review of such abnormalgrowth conditions, see Robbins and Angell, 1976, Basic Pathology, 2dEd., W. B. Saunders Co., Philadelphia, pp. 68-79.) Hyperplasia is a formof controlled cell proliferation involving an increase in cell number ina tissue or organ, without significant alteration in structure orfunction. As but one example, endometrial hyperplasia often precedesendometrial cancer. Metaplasia is a form of controlled cell growth inwhich one type of adult or fully differentiated cell substitutes foranother type of adult cell. Metaplasia can occur in epithelial orconnective tissue cells. Atypical metaplasia involves a somewhatdisorderly metaplastic epithelium. Dysplasia is frequently a forerunnerof cancer, and is found mainly in the epithelia; it is the mostdisorderly form of non-neoplastic cell growth, involving a loss inindividual cell uniformity and in the architectural orientation ofcells. Dysplastic cells often have abnormally large, deeply stainednuclei, and exhibit pleomorphism. Dysplasia characteristically occurswhere there exists chronic irritation or inflammation, and is oftenfound in the cervix, respiratory passages, oral cavity, and gallbladder.

The neoplastic lesion may evolve clonally and develop an increasingcapacity for invasion, growth, metastasis, and heterogeneity, especiallyunder conditions in which the neoplastic cells escape the host's immunesurveillance (Roitt, I., Brostoff, J and Kale, D., 1993, Immunology, 3rded., Mosby, St. Louis, pps. 17.1-17.12).

2.3. Immunotherapy

Four basic cell types whose function has been associated with antitumorcell immunity and the elimination of tumor cells from the body are: i)B-lymphocytes which secrete immunoglobulins into the blood plasma foridentifying and labeling the nonself invader cells; ii) monocytes whichsecrete the complement proteins which are responsible for lysing andprocessing the immunoglobulin-coated target invader cells; iii) naturalkiller lymphocytes having two mechanisms for the destruction of tumorcells-antibody-dependent cellular cytotoxicity and natural killing; andiv) T-lymphocytes possessing antigen-specific receptors and eachT-lymphocyte clone having the capacity to recognize a tumor cellcarrying complementary marker molecules (Schreiber, H., 1989, inFundamental Immunology (ed). W. E. Paul, pp. 923-955).

Several factors can influence the immunogenicity of tumors induced.These factors include dose of carcinogen, immunocompetence of the host,and latency period. Immunocompetence of the host during the period ofcancer induction and development can allow the host to respond toimmunogenic tumor cells. This may prevent the outgrowth of these cellsor select far less immunogenic escape variants that have lost theirrespective rejection antigen. Conversely, immunosuppression or immunedeficiency of the host during carcinogenesis or tumorigenesis may allowgrowth of highly immunogenic tumors (Schreiber, H., 1989, in FundamentalImmunology (ed). W. E. Paul, pp. 923-955).

Three major types of cancer immunotherapy are currently being explored:i) adoptive cellular immunotherapy, ii) in vivo manipulation of patientplasma to remove blocking factors or add tumoricidal factors, and iii)in vivo administration of biological response modifiers (e.g.,interferons (IFN; IFN-alpha and IFN-gamma), interleukins (IL; IL-2, IL-4and IL-6), colony-stimulating factors, tumor necrosis factor (TNF),monoclonal antibodies and other immunopotentiating agents, such ascorynebacterium parvum (C. parvum) (Kopp, W. C., et al., 1994, CancerChemotherapy and Biol. Response Modifiers 15:226-286). There is littledoubt that immunotherapy of cancer as it stands is falling short of thehopes invested in it. Although numerous immunotherapeutic approacheshave been tested, few of these procedures have proved to be effective asthe sole or even as an adjunct form of cancer prevention and treatment.

2.3.1. Adoptive Cellular Immunotherapy

Adoptive immunotherapy of cancer refers to a therapeutic approach inwhich immune cells with an antitumor reactivity are administered to atumor-bearing host, with the aim that the cells mediate either directlyor indirectly, the regression of an established tumor. Transfusion oflymphocytes, particularly T lymphocytes, falls into this category andinvestigators at the National Cancer Institute (NCI) have usedautologous reinfusion of peripheral blood lymphocytes ortumor-infiltrating lymphocytes (TIL), T cell cultures from biopsies ofsubcutaneous lymph nodules, to treat several human cancers (Rosenberg,S. A., U.S. Pat. No. 4,690,914, issued Sep. 1, 1987; Rosenberg, S. A.,et al., 1988, N. England J. Med. 319:1676-1680). For example, TILexpanded in vitro in the presence of interleukin (IL)-2 have beenadoptively transferred to cancer patients, resulting in tumor regressionin select patients with metastatic melanoma. Melanoma TIL grown in IL-2have been identified as activated T lymphocytes CD3⁺HLA-DR⁺, which arepredominantly CD8⁺ cells with unique in vitro antitumor properties. Manylong-term melanoma TIL cultures lyse autologous tumors in a specific MHCclass I- and T cell antigen receptor dependent manner (Topalian, S. L.,et al., 1989, J. Immunol. 142:3714). However, studies of TIL derivedfrom other types of tumors have revealed only scant evidence forcytolytic or proliferative antitumor immune specificity (Topalian, S. L.et al., 1990, in Important Advances in Oncology, V. T. DeVita, S. A.Hellman and S. A. Rosenberg, eds. J. B. Lippincott, Philadelphia, pp.19-41). In addition, the toxicity of the high-dose IL-2+activatedlymphocyte treatment advocated by the NCI group has been considerable,including high fevers, severe rigors, hypotension, damage to theendothelial wall due to capillary leak syndrome, and various adversecardiac events such..as arrhythmias and myocardial infarction (RosenbergS. A., et al., 1988, N. England J. Med. 319:1676-1680).

2.3.2. Interleukins (IL-2, IL-4 and IL-6)

IL-2 has significant antitumor activity in a small percentage ofpatients with renal cell carcinoma and melanoma. Investigators continueto search for IL-2 based regimens that will increase the response ratesin IL-2 responsive tumors, but, for the most part, have neither definednew indications nor settled fundamental issues, such as whether doseintensity is important in IL-2 therapy (Kopp, W. C., et al., 1994,Cancer Chemotherapy and Biol. Response Modifiers 15:226-286). Numerousreports have documented IL-2 associated toxicity involving increasednitrate levels and the syndrome of vascular leak and hypotension,analogous to septic shock. In addition, an increased incidence ofnonopportunistic bacterial infections and autoimmune complications arefrequently accompanied by the antitumor response of IL-2 (Kopp, W. C.,et al., 1994, Cancer Chemotherapy and Biol. Response Modifiers15:226-286).

IL-4 and IL-6 are also being tested as antitumor agents either directlyor through immunomodulating mechanisms. Dose-limiting toxicities havebeen observed with both agents in Phase I clinical trials (Gilleece, M.H., et al., 1992, Br. J. Cancer 66:204-210, Weber, J., et al., 1993, J.Clin. Oncol. 11:499-506).

2.3.3. Tumor Necrosis Factor

The toxicity of systemically administered TNF seriously limits its usefor the treatment of cancer. TNF has been most effective when used forregional therapy, in which measures, such as limb isolation forperfusion, are taken to limit the systemic dose and hence the toxicityof TNF. Dose-limiting toxicity of TNF consist of thrombocytopenia,headache, confusion and hypotension (Mittleman, A., et al., 1992, Inv.New Drugs 10:183-190).

2.3.4. Interferons

The activity of IFN-α has been described as being modest in a number ofmalignancies, including renal cell carcinoma, melanoma, hairy cellleukemia low-grade non-Hodgkin's lymphoma, and others. Higher doses ofIFN-α are usually associated with higher response rates in somemalignancies, but also cause more toxicity. In addition, more and morereports indicate that relapses after successful interferon therapycoincide with formation of neutralizing antibodies against interferon(Ouesada, J. R., et al., 1987, J. Interferon Res. 67:678.

2.4. Pharmacokinetic Models for Anticancer Chemotherapeutic andImmunotherapeutic Drugs: Extrapolation and Scaling of Animal Data toHumans

The ethical and fiscal constraints which require the use of animalmodels for most toxicology research also impose the acceptance ofcertain fundamental assumptions in order to estimate dose potency inhumans from dose-response data in animals. Interspecies dose-responseequivalence is most frequently estimated as the product of a referencespecies dose and a single scaling ratio based on a physiologicalparameter such as body weight, body surface area, maximum lifespanpotential, etc. Most frequently, exposure is expressed as milligrams ofdose administered in proportion to body mass in kilograms (mg kg⁻¹).Body mass is a surrogate for body volume, and therefore, the ratiomilligrams per kilogram is actually concentrations in milligrams perliter (Hirshaut, Y., et al., 1969, Cancer Res. 29:1732-1740). The keyassumptions which accompany this practice and contribute to its failureto accurately estimate equipotent exposure among various species are: i)that the biological systems involved are homogeneous, “well-stirredvolumes” with specific gravity equal to 1.0; ii) that the administeredcompounds are instantly and homogeneously distributed throughout thetotal body mass; and iii) that the response of the biological systems isdirectly proportional only to the initial concentration of the testmaterial in the system. As actual pharmacokinetic conditions :departfrom these assumptions, the utility of initial concentration scalingbetween species declines.

Through pharmacokinetics, one can study the time course of a drug andits metabolite levels in different fluids, tissues, and excreta of thebody, and the mathematical relationships required to develop models tointerpret such data. It, therefore, provides the basic informationregarding drug distribution, availability, and the resulting toxicity inthe tissues and hence, specifies the limitation in the drug dosage fordifferent treatment schedules and different routes of drugadministration. The ultimate goal of the pharmacokinetic studies ofanticancer drugs is thus to offer a framework for the design of optimaltherapeutic dosage regimens and treatment schedules for individualpatients.

The currently utilized guidelines for prescription have evolvedgradually without always having a complete and explicit justification.In 1966, Freireich and co-workers proposed the use of surface areaproportions for interspecies extrapolation of the acute toxicity ofanticancer drugs. This procedure has become the method of choice formany risk assessment applications (Freireich, E. J., et al., 1966,Cancer Chemotherapy Rep. 50:219-244). For example, surface area scalingis the basis of the National Cancer Institute's interspeciesextrapolation procedure for anti-cancer drugs (Schein, P. S., et al.,1970, Clin. Pharmacol. Therap. 11:3-40; Goldsmith, M. A., et al., 1975,Cancer Res. 35:1354-1364). In accepting surface area extrapolation, thetenuous basis for initial concentration scaling has been replaced by anempirical approach. The basic formula used for estimating prescriptionof cancer chemotherapy per body surface area (BSA) is BSA=k×kg^(2/3), inwhich k is a constant that differs for each age group and species. Forexample, the k value for adult humans is 11, while for mice it is 9 (SeeQuiring, P., 1955, Surface area determination, in Glasser E. (ed.)Medical Physics I Chicago: Medical Year Book, p. 1490 and Vriesendorp,H. M., 1985, Hematol. (Supplm. 16) 13:57-63). The major attraction ofexpressing cancer chemotherapy per m² BSA appears to be that it offersan easily remembered simplification, i.e., equal doses of drug per m²BSA will produce approximately the same effect in comparing differentspecies and age groups. However, simplicity is not proof and alternativemethods for estimating prescription of anticancer drugs appear to have abetter scientific foundation, with the added potential for a moreeffective use of anticancer agents (Hill, J.A., et al., 1989, HealthPhysics 57:395-401).

The effectiveness of an optimal dose of a drug used in chemotherapyand/or immunotherapy can be altered by various factors, including tumorgrowth kinetics, drug resistance of tumor cells, total-body tumor cellburden, toxic effects of chemotherapy and/or immunotherapy on cells andtissues other than the tumor, and distribution of chemotherapeuticagents and/or immunotherapeutic agents within the tissues of thepatient. The greater the size of the primary tumor, the greater theprobability that a large number of cells (drug resistant and drugsensitive) have metastasized before diagnosis and that the patient willrelapse after the primary.

Some metastases arise in certain sites in the body where resistance tochemotherapy is based on the limited tissue distribution ofchemotherapeutic drugs administered in standard doses. Such sites act assanctuaries that shield the cancer cells from drugs that are circulatingin the blood; for example, there are barriers in the brain and testesthat impede drug diffusion from the capillaries into the tissue. Thus,these sites may require special forms of treatment such asimmunotherapy, especially since immunosuppression is characteristic ofseveral types of neoplastic diseases.

3. SUMMARY OF THE INVENTION

The methods of the invention comprise methods of eliciting an immuneresponse in an individual in whom the treatment or prevention of canceror infectious disease is desired by administering, preferablyintradermally or mucosally, a composition comprising an effective amountof a complex in which the complex consists essentially of heat shockprotein(s) (hsp(s)) noncovalently bound to antigenic molecule(s). Theamounts of the complex that are administered are within ranges ofeffective dosages, discovered by the present inventor to be effective,and which are surprisingly smaller than those amounts predicted to beeffective by extrapolation by prior art methods from dosages used inanimal studies. In a preferred embodiment, the complex is autologous tothe individual; that is, the complex is isolated from the cancer cellsof the individual himself (e.g., preferably prepared from tumor biopsiesof the patient). Alternatively, the hsp and or the antigenic moleculecan be isolated from the individual or from others or by recombinantproduction methods using a cloned hsp originally derived from theindividual or from others. “Antigenic molecule” as used herein refers tothe peptides with which the hsps are endogenously associated in vivo(e.g., in precancerous or cancerous tissue), as well as exogenousantigens/immunogens (i.e., with which the hsps are not complexed invivo) or antigenic/immunogenic fragments and derivatives thereof. Suchexogenous antigens and fragments and derivatives (both peptide andnon-peptide) thereof for use in complexing with hsps, can be selectedfrom among those known in the art, as well as those readily identifiedby standard immunoassays known in the art by detecting the ability tobind antibody or MHC molecules (antigenicity) or generate immuneresponse (immunogenicity).

In the practice of the invention, therapy by administration ofhsp-peptide complexes using any convenient route of administration mayoptionally be in combination with adoptive immunotherapy involving theadministration of antigen-presenting cells that have been sensitized invitro with complexes of hsp(s) noncovalently bound to antigenicmolecules. The methods for adoptive immunotherapy of cancer andinfectious diseases have the goal of enhancing the host'simmunocompetence and activity of immune effector cells. Adoptiveimmunotherapy with macrophages and/or other antigen-presenting cells(APC), for example, dendritic cells and B cells (B lymphocytes), thathave been sensitized in vitro with noncovalent complexes of an hspnoncovalently bound to an antigenic molecule, induces specific immunityto tumor cells and/or antigenic components, promoting regression of thetumor mass or treatment of immunological disorders or infectiousdiseases, as the case may be.

In a specific embodiment, the present invention relates to methods andcompositions for prevention and treatment of primary and metastaticneoplastic diseases.

Specific therapeutic regimens, pharmaceutical compositions, and kits areprovided by the invention. In contrast to the prior art, the dosages ofthe hsp-antigenic molecule complex are not based on, and are smallerthan those dosages based on, body weight or surface area of the patient.The present inventor has discovered that a dosage substantiallyequivalent to or smaller than that seen to be effective in smallernon-human mammals (e.g., mice) is effective for human intradermaladministration, optionally subject to a correction factor not exceedinga fifty fold increase, based on the relative lymph node sizes in suchmammals and in humans. The present inventor has discovered thateffective intradermal dosages are about tenfold smaller even than thesurprisingly small doses effective in subcutaneous administration inhumans. (see U.S. Pat. No. 5,837,251, which is incorporated by referenceherein in its entirety.) Pharmaceutical formulations are provided, basedon these newly-discovered effective dose ranges for humans, comprisingcompositions of complexes of antigenic molecules and heat shock/stressproteins, including but not limited to hsp70 , hsp90, , gp96 eitheralone or in combination. Specifically, interspecies dose-responseequivalence for hsp noncovalently bound to antigenic molecules for ahuman intradermal or mucosal dose is estimated as the product of thetherapeutic dosage observed in mice and a single scaling ratio, notexceeding a fifty fold increase.

The present invention encompasses methods for prevention and treatmentof cancer by enhancing the host's immune competence and activity ofimmune effector cells. Furthermore, the invention provides methods forevaluating the efficacy of drugs in enhancing immune responses fortreatment and monitoring the progress of patients participating inclinical trials for the treatment of primary and metastatic neoplasticdiseases.

Immunotherapy using the therapeutic regimens of the invention, byadministering such complexes of heat shock/stress proteins noncovalentlybound to antigenic molecules, can induce specific immunity to tumorcells, and leads to regression of the tumor mass. Cancers which areresponsive to specific immunotherapy by administering the heatshock/stress proteins of the invention include but are not limited tohuman sarcomas and carcinomas. In a specific embodiment, thehsp-antigenic molecule complexes are allogeneic to the patient; in apreferred embodiment, the hsp-antigenic molecule complexes areautologous to (derived from) the patient to whom they are administered.

Particular compositions of the invention and their properties aredescribed in the sections and subsections which follow. A preferredcomposition comprises hsp-peptide complexes isolated from the tumorbiopsy of the patient to whom the composition is to be administered.Such a composition that comprises hsp70 , hsp90, and/or gp96demonstrates strong inhibition of a variety of tumors in mammals.Moreover, the therapeutic doses that are effective in the correspondingexperimental model in rodents as described infra, in Section 6 can beused to inhibit the in vivo growth of colon and liver cancers in humancancer patients as described in Sections 7 and 8, infra. Preferredcompositions comprising hsp70, , hsp90, and/or gp96 which preferablyexhibit no toxicity when administered to human subjects are alsodescribed.

In another embodiment, the methods further optionally compriseadministering biological response modifiers, e.g., IFN-α, IFN-γ, IL-2,IL-4, IL-6, TNF, or other cytokine growth factors affecting the immunecells, in combination with the hsp complexes.

In addition to cancer therapy, the complexes of hsps noncovalently boundto antigenic molecules can be utilized for the prevention of a varietyof cancers, e.g., in individuals who are predisposed as a result offamilial history or in individuals with an enhanced risk to cancer dueto environmental factors.

The Examples presented in Sections 6, 7 and 8 below, detail the useaccording to the methods of the invention of hsp-peptide complexes incancer immunotherapy in experimental tumor models and in human patientssuffering from advanced colon and liver cancer.

4. BRIEF DESCRIPTION OF FIGURES

FIGS. 1A-C. Effect of intradermal administration of gp96 on retardationof tumor growth measured as average tumor diameter (mm).

FIG. 1A: Mice were injected intradermally in different sites with buffersolution, twice at weekly intervals. One week after the secondinjection, the mice were challenged with 1×10⁵ Meth A sarcoma cells.

FIG. 1B: Mice were injected intradermally in different sites with 1microgram of gp96-antigenic molecule complex derived from Meth A sarcomacells, twice at weekly intervals. One week after the second injection,the mice were challenged with 1×10⁵ Meth A sarcoma cells.

FIG. 1C: Mice were injected intradermally in different sites with 5micrograms of gp96-antigenic molecule complex derived from Meth Asarcoma cells, twice at weekly intervals. One week after the secondinjection, the mice were challenged with 1×10⁵ Meth A sarcoma cells.

5. DETAILED DESCRIPTION OF THE INVENTION

Methods and compositions for the prevention and treatment of primary andmetastatic neoplastic diseases and infectious diseases and for elicitingan immune response in a human individual, are described. The inventionis based, in part, on a newly discovered dosage regimen foradministration of compositions comprising complexes of hspsnoncovalently bound to antigenic molecules. The present inventor hasdiscovered that a dosage substantially equivalent to or smaller thanthat seen to be effective in smaller non-human animals (e.g., mice) iseffective for human intradermal administration, such as described inSection 5.1, below.

“Antigenic molecule” as used herein refers to the peptides with whichthe hsps are endogenously associated in vivo (e.g., in infected cells orprecancerous or cancerous tissue) as well as exogenousantigens/immunogens (i.e., with which the hsps are not complexed invivo) or antigenic/immunogenic fragments and derivatives thereof.

The methods of the invention comprise methods of eliciting an immuneresponse in an individual in whom the treatment or prevention ofinfectious diseases or cancer is desired by administering, preferablyintradermally or mucosally, a composition comprising an effective amountof a complex, in which the complex consists essentially of an hspnoncovalently bound to an antigenic molecule.

In the practice of the invention, therapy by administration ofhsp-antigenic molecule complexes using any convenient mode ofadministration may optionally be in combination with adoptiveimmunotherapy. The APC can be selected from among those antigenpresenting cells known in the art, including but not limited tomacrophages, dendritic cells, B lymphocytes, and a combination thereof,and are preferably macrophages. The hsp-antigenic molecule-ensitized APCmay be administered concurrently or before or after administration ofthe hsp-antigenic molecule complexes. The hsp-antigenic molecule complexthat is administered to the patient can be the same or different fromthe hsp-antigenic molecule complex used to sensitize the APC that areadministered to the patient. In a specific embodiment wherein the APCand hsp-antigenic molecule complexes are administered concurrently, theAPC and hsp-antigenic molecule complexes can be present in a singlecomposition or different composition for administration. Adoptiveimmunotherapy according to the invention allows activation of immuneantigen presenting cells by incubation with hsp-antigenic moleculecomplexes. Preferably, prior to use of the cells in vivo measurement ofreactivity against the tumor or infectious agent in vitro is done. Thisin vitro boost followed by clonal selection and/or expansion, andpatient administration constitutes a useful therapeutic/prophylacticstrategy.

In a preferred embodiment, the hsp-antigenic molecule complex isautologous to the individual; that is, the complex is isolated fromeither the infected cells or the cancer cells or precancerous cells ofthe individual himself (e.g., preferably prepared from infected tissuesor tumor biopsies of the patient). Alternatively, the complex isproduced in vitro (e.g., wherein a complex with an exogenous antigenicmolecule is desired). Alternatively, the hsp and/or the antigenicmolecule can be isolated from the individual or from others or made byrecombinant production methods using a cloned hsp originally derivedfrom the individual or from others. Exogenous antigens and fragments andderivatives (both peptide and non-peptide) thereof for use in complexingwith hsps, can be selected from among those known in the art, as well asthose readily identified by standard immunoassays known in the art bythe ability to bind antibody or MHC molecules (antigenicity) or generateimmune response (immunogenicity). Complexes of hsps and antigenicmolecules can be isolated from cancer or precancerous tissue of apatient, or from a cancer cell line, or can be produced in vitro (as isnecessary in the embodiment in which an exogenous antigen is used as theantigenic molecule).

The hsps of the present invention that can be used include but are notlimited to, hsp70 , hsp90, , gp96 alone or in combination. Preferably,the hsps are human hsps.

Heat shock proteins, which are also referred to interchangeably hereinas stress proteins, useful in the practice of the instant invention canbe selected from among any cellular protein that satisfies any one ofthe following criteria. It is a protein whose intracellularconcentration increases when a cell is exposed to a stressful stimuli,it is capable of binding other proteins or peptides, it is capable ofreleasing the bound proteins or peptides in the presence of adenosinetriphosphate (ATP) or low pH, or it is a protein showing at least 35%homology with any cellular protein having any of the above properties.

The first stress proteins to be identified were the heat shock proteins(hsps). As their name implies, hsps are synthesized by a cell inresponse to heat shock. To date, three major families of hsp have beenidentified based on molecular weight. The families have been calledhsp60, , hsp70 and hsp90, where the numbers reflect the approximatemolecular weight of the stress proteins in kilodaltons. Many members ofthese families were found subsequently to be induced in response toother stressful stimuli including, but not limited to, nutrientdeprivation, metabolic disruption, oxygen radicals, and infection withintracellular pathogens. (See Welch, May 1993, Scientific American56-64; Young, 1990, Annu. Rev. Immunol. 8:401-420; Craig, 1993, Science260:1902-1903; Gething, et al., 1992, Nature 355:33-45; and Lindquist,et al., 1988, Annu. Rev. Genetics 22:631-677), the disclosures of whichare incorporated herein by reference. It is contemplated thathsps/stress proteins belonging to all of these three families can beused in the practice of the instant invention.

The major hsps can accumulate to very high levels in stressed cells, butthey occur at low to moderate levels in cells that have not beenstressed. For example, the highly inducible mammalian hsp70 is hardlydetectable at normal temperatures but becomes one of the most activelysynthesized proteins in the cell upon heat shock (Welch, et al., 1985,J. Cell. Biol. 101:1198-1211). In contrast, hsp90, and hsp60, proteinsare abundant at normal temperatures in most, but not all, mammaliancells and are further induced by heat (Lai, et al., 1984, Mol. Cell.Biol. 4:2802-10; van Bergen en Henegouwen, et al., 1987, Genes Dev.1:525-31).

Heat shock proteins are among the most highly conserved proteins inexistence. For example, DnaK, the hsp70 from E. coli has about 50% aminoacid sequence identity with hsp70 proteins from excoriates (Bardwell, etal., 1984, Proc. Natl. Acad. Sci. 81:848-852). The hsp60, and hsp90,families also show similarly high levels of intrafamilies conservation(Hickey, et al., 1989, Mol. Cell. Biol. 9:2615-2626; Jindal, 1989, Mol.Cell. Biol. 9:2279-2283). In addition, it has been discovered that thehsp60, , hsp70 and hsp90, families are composed of proteins that arerelated to the stress proteins in sequence, for example, having greaterthan 35% amino acid identity, but whose expression levels are notaltered by stress. Therefore it is contemplated that the definition ofheat shock protein or stress protein, as used herein, embraces otherproteins, muteins, analogs, and variants thereof having at least 35% to55%, preferably 55% to 75%, and most preferably 75% to 85% amino acididentity with members of the three families whose expression levels in acell are enhanced in response to a stressful stimulus. The purificationof stress proteins belonging to these three families is described below.

The immunogenic hsp-peptide complexes of the invention may include anycomplex containing an hsp and a peptide that is capable of inducing animmune response in a mammal. The peptides are preferably noncovalentlyassociated with the hsp. Preferred complexes may include, but are notlimited to, hsp60-peptide, hsp70-peptide and hsp90-peptide complexes.For example, an hsp called gp96 which is present in the endoplasmicreticulum of eukaryotic cells and is related to the cytoplasmic hsp90'scan be used to generate an effective vaccine containing a gp96-peptidecomplex.

Although the hsps can be allogeneic to the patient, in a preferredembodiment, the hsps are autologous to (derived from) the patient towhom they are administered. The hsps and/or antigenic molecules can bepurified from natural sources, chemically synthesized, or recombinantlyproduced.

The invention provides combinations of compositions which enhance theimmunocompetence of the host individual and elicit specific immunityagainst infectious agents or specific immunity against preneoplastic andneoplastic cells. The therapeutic regimens and pharmaceuticalcompositions of the invention are described below. These compositionshave the capacity to prevent the onset and progression of infectiousdiseases and prevent the development of tumor cells and to inhibit thegrowth and progression of tumor cells indicating that such compositionscan induce specific immunity in infectious diseases and cancerimmunotherapy.

Hsps appear to induce an inflammatory reaction at the tumor site andultimately cause a regression of the tumor burden in the cancer patientstreated. Cancers which can be treated with complexes of hspsnoncovalently bound to antigenic molecules include, but are not limitedto, human sarcomas and carcinomas. Human sarcomas and carcinomas arealso responsive to adoptive immunotherapy by the hsp complex-sensitizedmacrophages and/or APC.

Accordingly, the invention provides methods of preventing and treatingcancer in an individual comprising administering hsp-antigenic moleculecomplexes, optionally in combination with APC sensitized by suchcomplexes, which stimulates the immunocompetence of the host individualand elicits specific immunity against the preneoplastic and/orneoplastic cells. As used herein, “preneoplastic” cell refers to a cellwhich is in transition from a normal to a neoplastic form; andmorphological evidence, increasingly supported by molecular biologicstudies, indicates that preneoplasia progresses through multiple steps.Non- neoplastic cell growth commonly consists of hyperplasia,metaplasia, or most particularly, dysplasia (for review of such abnormalgrowth conditions (See Robbins and Angell, 1976, Basic Pathology, 2dEd., W. B. Saunders Co., Philadelphia, pp. 68-79). Hyperplasia is a formof controlled cell proliferation involving an increase in cell number ina tissue or organ, without significant alteration in structure orfunction. As but one example, endometrial hyperplasia often precedesendometrial cancer. Metaplasia is a form of controlled cell growth inwhich one type of adult or fully differentiated cell substitutes foranother type of adult cell. Metaplasia can occur in epithelial orconnective tissue cells. Atypical metaplasia involves a somewhatdisorderly metaplastic epithelium. Dysplasia is frequently a forerunnerof cancer, and is found mainly in the epithelia; it is the mostdisorderly form of non-neoplastic cell growth, involving a loss inindividual cell uniformity and in the architectural orientation ofcells. Dysplastic cells often have abnormally large, deeply stainednuclei, and exhibit pleomorphism. Dysplasia characteristically occurswhere there exists chronic irritation or inflammation, and is oftenfound in the cervix, respiratory passages, oral cavity, and gallbladder. Although preneoplastic lesions may progress to neoplasia, theymay also remain stable for long periods and may even regress,particularly if the inciting agent is removed or if the lesion succumbsto an immunological attack by its host.

The therapeutic regimens and pharmaceutical compositions of theinvention may be used with additional immune response enhancers orbiological response modifiers including, but not limited to, thecytokines IFN-α, IFN-γ, IL-2, IL-4, IL-6, TNF, or other cytokineaffecting immune cells. In accordance with this aspect of the invention,the complexes of the hsp and antigenic molecule are administered incombination therapy with one or more of these cytokines.

The invention further relates to administration of complexes ofhsp-antigenic molecules, optionally in combination with APC sensitizedby such complexes, to individuals at enhanced risk of cancer due tofamilial history or environmental risk factors.

5.1. Dosage Regimens

It was established in experimental tumor models (Blachere et al., 1993,J. Immunotherapy 14:352-356) that the lowest dose of hsp noncovalentlybound to peptide complexes which produced tumor regression in mice wasbetween 10 and 25 microgram/mouse weighing 20-25 g which is equal to 25μg/25 g=1 mg/kg. Prior art methods extrapolate to human dosages based onbody weight and surface area. For example, prior art methods ofextrapolating human dosage based on body weight can be carried out asfollows: since the conversion factor for converting the mouse dosage tohuman dosage is Dose Human per kg=Dose Mouse per kg×12 (See Freireich,E. J., et al., 1966, Cancer Chemotherap. Rep. 50:219-244), the effectivedose of hsp-peptide complexes in humans weighing 70 kg should belmg/kg÷12×70, i.e., about 6 mg (5.8 mg).

Drug doses are also given in milligrams per square meter of body surfacearea because this method rather than body weight achieves a goodcorrelation to certain metabolic and excretionary functions (Shirkey, H.C., 1965, JAMA 193:443). Moreover, body surface area can be used as acommon denominator for drug dosage in adults and children as well as indifferent animal species as indicated below in Table 1 (Freireich, E.J., et al., 1966, Cancer Chemotherap. Rep. 50:219-244).

TABLE 1 REPRESENTATIVE SURFACE AREA TO WEIGHT RATIOS (km) FOR VARIOUSSPECIES¹ Species Body Weight (kg) Surface Area (Sq m) km Factor Mouse0.02 0.0066 3.0 Rat 0.15 0.025 5.9 Monkey 3.0 0.24 12 Dog 8.0 0.40 20Human, Child 20 0.80 25 Adult 60 1.6 37 Example: To express a mg/kg dosein any given species as the equivalent mg/sq m dose, multiply the doseby the appropriate km factor. In an adult human, 100 mg/kg is equivalentto 100 mg/kg × 37 kg/sq m = 3700 mg/sq m. ¹Freireich, et al., 1966,Cancer Chemotherap. Rep. 50: 219-244.

In contrast to both of the above-described prior art methods ofdetermining dosage levels, the present invention provides dosages of thepurified complexes of hsps and antigenic molecules that are much smallerthan the dosages estimated by prior art methods. For example, accordingto a preferred embodiment of the invention, an amount of hsp70- and/orgp96-antigenic molecule complexes is administered that is in the rangeof about 0.1 micrograms to about 60 micrograms for a human patient. Inanother specific embodiment, the therapeutically effective amount ofhsp70- and/or gp96-antigenic molecule complexes is less than 10micrograms, e.g., in the range of 0.1 to 9 micrograms; the preferredhuman dosage being substantially equivalent to or smaller than thedosage used in a 25 g mouse, e.g., in the range of 0.5 to 2.0micrograms. The preferred dosage for hsp90-antigenic molecule complexesin a human patient provided by the present invention is in the range ofabout 5 to 500 micrograms. In a specific embodiment, the therapeuticallyeffective amount of hsp90-antigenic molecule complexes is less than 50micrograms, e.g., in the range of 5 to 49 micrograms; the preferreddosage being in the range of 5 to 40 micrograms.

The doses recited above are preferably administered intradermally ormucosally. By way of example, the doses can be administered, preferablyintradermally, every other day for a total of 5 injections. In apreferred embodiment, the doses recited above are given once weekly fora period of about 4 to 6 weeks, and the mode of site of administrationis preferably varied with each administration. In a preferred example,intradermal administrations are given, with each site of administrationvaried sequentially. Thus, by way of example and-not limitation, thefirst injection may be given intradermally on the left arm, the secondon the right arm, the third on the left belly, the fourth on the rightbelly, the fifth on the left thigh, the sixth on the right thigh, etc.The same site may be repeated after a gap of one or more injections.Also, split injections may be given. Thus, for example, half the dosemay be given in one site and the other half in another site on the sameday.

After 4-6 weeks, further injections are preferably given at two-weekintervals over a period of time of one month. Later injections may begiven monthly. The pace of later injections may be modified, dependingupon the patient's clinical progress and responsiveness to theimmunotherapy. Alternatively, the mode of administration is sequentiallyvaried, e.g., weekly administrations are given in sequence intradermallyor mucosally.

In an embodiment wherein adoptive immunotherapy is also employed, theabove regimens for administration of hsp-antigenic molecule complexesmay occur before, during or after administration of the hsp-antigenmolecule-sensitized APC. For example, the mode of therapy issequentially varied, e.g., hsp-antigenic molecule complexes may beadministered at one time and hsp-antigenic molecule-sensitized APCanother time. Preferably the hsp-antigenic molecule-sensitized APC andthe hsp-antigenic molecule complexes are administered to the patientwithin 1 week of each other.

The invention is illustrated by non-limiting examples in Sections 6, 7and 8.

5.2. Therapeutic Compositions Comprising Purified Hsp-Peptide Complexes,for Eliciting Immune Responses to Cancer or Infectious Disease, and forIn Vitro Sensitization of APC

The compositions comprising hsp noncovalently bound to antigenicmolecules are administered to elicit an effective specific immuneresponse to the complexed antigenic molecules (and not to the hsp). Inaccordance with the methods described herein, the hsp-antigenic moleculecomplexes are preferably purified in the range of 60 to 100 percent ofthe total mg protein, or at least 70%, 80% or 90% of the total mgprotein. In another embodiment, the hsp-antigenic molecule complexes arepurified to apparent homogeneity, as assayed by sodium dodecylsulfate-polyacrylamide gel electrophoresis.

In a preferred embodiment, non-covalent complexes of hsp70 , hsp90, andgp96 with peptides are prepared and purified postoperatively from tumorcells obtained from the cancer patient.

In accordance with the methods described herein, immunogenic orantigenic peptides that are endogenously complexed to hsps or MHCantigens can be used as antigenic molecules. For example, such peptidesmay be prepared that stimulate cytotoxic T cell responses againstdifferent tumor antigens (e.g., tyrosinase, gp100, melan-A, gp75,mucins, etc.) and viral proteins including, but not limited to, proteinsof immunodeficiency virus type I (HIV-I), human immunodeficiency virustype II (HIV-II) , hepatitis type A, hepatitis type B, hepatitis type C,influenza, Varicella, adenovirus, herpes simplex type I (HSV-I), herpessimplex type II (HSV-II), rinderpest, rhinovirus, echovirus, rotavirus,respiratory syncytial virus, papilloma virus, papova virus,cytomegalovirus, echinovirus, arbovirus, huntavirus, coxsackie virus,mumps virus, measles virus, rubella virus and polio virus. In theembodiment wherein the antigenic molecules are peptides noncovalentlycomplexed to hsps in vivo, the complexes can be isolated from cells, oralternatively, produced in vitro from purified preparations each of hspsand antigenic molecules.

In another specific embodiment, antigens of cancers (e.g., tumors) orinfectious agents (e.g., viral antigen, bacterial antigens, etc.) can beobtained by purification from natural sources, by chemical synthesis, orrecombinantly, and, through in vitro procedures such as that describedbelow, noncovalently complexed to hsps.

In an embodiment wherein the hsp-antigenic molecule complex to be usedis a complex that is produced in vivo in cells, exemplary purificationprocedures such as described in Sections 5.2.1-5.2.3 below can beemployed. Alternatively, in an embodiment wherein one wishes to useantigenic molecules by complexing to hsps in vitro, hsps can be purifiedfor such use from the endogenous hsp-peptide complexes in the presenceof ATP or low pH (or chemically synthesized or recombinantly produced).The protocols described herein may be used to isolate hsp-peptidecomplexes, or the hsps alone, from any eukaryotic cells for example,tissues, isolated cells, or immortalized eukaryote cell lines infectedwith a preselected intracellular pathogen, tumor cells or tumor celllines.

5.2.1. Preparation and Purification of Hsp70-peptide Complexes

The purification of hsp70 -peptide complexes has been describedpreviously, see, for example, Udono et al., 1993, J. Exp. Med.178:1391-1396. A procedure that may be used, presented by way of examplebut not limitation, is as follows:

Initially, tumor cells are suspended in 3 volumes of 1×Lysis bufferconsisting of 5 mM sodium phosphate buffer, pH 7, 150 mM NaCl, 2 mMCaCl₂,2 mM MgCl₂ and 1 mM phenyl methyl sulfonyl fluoride (PMSF). Then,the pellet is sonicated, on ice, until >99% cells are lysed asdetermined by microscopic examination. As an alternative to sonication,the cells may be lysed by mechanical shearing and in this approach thecells typically are resuspended in 30 mM sodium bicarbonate pH 7.5, 1 mMPMSF, incubated on ice for 20 minutes and then homogenized in a Douncehomogenizer until >95% cells are lysed.

Then the lysate is centrifuged at 1,000 g for 10 minutes to removeunbroken cells, nuclei and other cellular debris. The resultingsupernatant is recentrifuged at 100,000 g for 90 minutes, thesupernatant harvested and then mixed with Con A Sepharose equilibratedwith phosphate buffered saline (PBS) containing 2 mM Ca²⁺ and 2 mM Mg²⁺.When the cells are lysed by mechanical shearing the supernatant isdiluted with an equal volume of 2×lysis buffer prior to mixing with ConA Sepharose. The supernatant is then allowed to bind to the Con ASepharose for 2-3 hours at 4° C. The material that fails to bind isharvested and dialyzed for 36 hours (three times, 100 volumes each time)against 10 mM Tris-Acetate pH 7.5, 0.1 mM EDTA, 10 mM NaCl, 1 mM PMSF.Then the dialyzate is centrifuged at 17,000 rpm (Sorvall SS34 rotor) for20 minutes. Then the resulting supernatant is harvested and applied to aMono Q FPLC column equilibrated in 20 mM Tris-Acetate pH 7.5, 20 mMNaCl, 0.1 mM EDTA and 15 mM 2-mercaptoethanol. The column is thendeveloped with a 20 mM to 500 mM NaCl gradient and then eluted fractionsfractionated by sodium dodecyl sulfate-polyacrylamide gelelectrophoresis (SDS-PAGE) and characterized by immunoblotting using anappropriate anti-hsp70 antibody (such as from clone N27F3-4, fromStressGen).

Fractions strongly immunoreactive with the anti-hsp70, antibody arepooled and the hsp70-peptide complexes precipitated with ammoniumsulfate; specifically with a 50%-70% ammonium sulfate cut. The resultingprecipitate is then harvested by centrifugation at 17,000 rpm (SS34Sorvall rotor) and washed with 70% ammonium sulfate. The washedprecipitate is then solubilized and any residual ammonium sulfateremoved by gel filtration on a Sephadex^(R) G25 column (Pharmacia). Ifnecessary the hsp70 preparation thus obtained can be repurified throughthe Mono Q FPLC Column as described above.

The hsp70-peptide complex can be purified to apparent homogeneity usingthis method. Typically 1 mg of hsp70-peptide complex can be purifiedfrom 1 g of cells/tissue.

An improved method for purification of hsp70-peptide complexes comprisescontacting cellular proteins with ADP or a nonhydrolyzable analog of ATPaffixed to a solid substrate, such that hsp70 in the lysate can bind tothe ADP or nonhydrolyzable ATP analog, and eluting the bound hsp70. Apreferred method uses column chromatography with ADP affixed to a solidsubstratum (e.g., ADP-agarose). The resulting hsp70 preparations arehigher in purity and devoid of contaminating peptides. The hsp70 yieldsare also increased significantly by about more than 10 fold.Alternatively, chromatography with nonhydrolyzable analogs of ATP,instead of ADP, can be used for purification of hsp70-peptide complexes.By way of example but not limitation, purification of hsp70-peptidecomplexes by ADP-agarose chromatography can be carried out as follows:

Meth A sarcoma cells (500 million cells) are homogenized in hypotonicbuffer and the lysate is centrifuged at 100,000 g for 90 minutes at 4°C. The supernatant is applied to an ADP-agarose column. The column iswashed in buffer and is eluted with 5 column volumes of 3 mM ADP. Thehsp70-peptide complexes elute in fractions 2 through 10 of the total 15fractions which elute. The eluted fractions are analyzed by SDS-PAGE.The hsp70-peptide complexes can be purified to apparent homogeneityusing this procedure.

5.2.2. Preparation and Purification of HsP90-peptide Complexes

A procedure that can be used, presented by way of example and notlimitation, is as follows:

Initially, tumor cells are suspended in 3 volumes of 1×Lysis bufferconsisting of 5 mM sodium phosphate buffer (pH7), 150 mM NaCl, 2 mMCaCl₂, 2 mM MgCl₂ and 1 mM phenyl methyl sulfonyl fluoride (PMSF). Then,the pellet is sonicated, on ice, until >99% cells are lysed asdetermined by microscopic examination. As an alternative to sonication,the cells may be lysed by mechanical shearing and in this approach thecells typically are resuspended in 30 mM sodium bicarbonate pH 7.5, 1 mMPMSF, incubated on ice for 20 minutes and then homogenized in a Douncehomogenizer until >95% cells are lysed.

Then the lysate is centrifuged at 1,000 g for 10 minutes to removeunbroken cells, nuclei and other cellular debris. The resultingsupernatant is recentrifuged at 100,000 g for 90 minutes, thesupernatant harvested and then mixed with Con A Sepharose equilibratedwith PBS containing 2 mM Ca²⁺ and 2 mM Mg^(2×). When the cells are lysedby mechanical shearing the supernatant is diluted with an equal volumeof 2×Lysis buffer prior to mixing with Con A Sepharose. The supernatantis then allowed to bind to the Con A Sepharose for 2-3 hours at 4° C.The material that fails to bind is harvested and dialyzed for 36 hours(three times, 100 volumes each time) against 10 mM Tris-Acetate pH 7.5,0.1 mM EDTA, 10 mM NaCl, 1 mM PMSF. Then the dialyzate is centrifuged at17,000 rpm (Sorvall SS34 rotor) for 20 minutes. Then the resultingsupernatant is harvested and applied to a Mono Q FPLC columnequilibrated with lysis buffer. The proteins are then eluted with a saltgradient of 200 mM to 600 mM NaCl.

The eluted fractions are fractionated by SDS-PAGE and fractionscontaining the hsp90-peptide complexes identified by immunoblottingusing an anti-hsp90, antibody such as 3G3 (Affinity Bioreagents).Hsp90-peptide complexes can be purified to apparent homogeneity usingthis procedure. Typically, 150-200 μg of hsp90-peptide complex can bepurified from 1 μg of cells/tissue.

5.2.3. Preparation and Purification of qp96-peptide Complexes

A procedure that can be used, presented by way of example and notlimitation, is as follows:

A pellet of tumors is resuspended in 3 volumes of buffer consisting of30 mM sodium bicarbonate buffer (pH 7.5) and 1 mM PMSF and the cellsallowed to swell on ice 20 minutes. The cell pellet is then homogenizedin a Dounce homogenizer (the appropriate clearance of the homogenizerwill vary according to each cell type) on ice until >95% cells arelysed.

The lysate is centrifuged at 1,000 g for 10 minutes to remove unbrokencells, nuclei and other debris. The supernatant from this centrifugationstep is then recentrifuged at 100,000 g for 90 minutes. The gp96-peptidecomplex can be purified either from the 100,000 pellet or from thesupernatant.

When purified from the supernatant, the supernatant is diluted withequal volume of 2×lysis buffer and the supernatant mixed for 2-3 hoursat 4° C. with Con A Sepharose equilibrated with PBS containing 2 mM Ca²⁺and 2 mM Mg²⁺. Then, the slurry is packed into a column and washed with1×lysis buffer until the OD₂₈₀ drops to baseline. Then, the column iswashed with 1/3 column bed volume of 10% α-methyl mannoside (α-MM)dissolved in PBS containing 2 mM Ca²⁺ and 2 mM Mg²⁺, the column sealedwith a piece of parafilm, and incubated at 37° C. for 15 minutes. Thenthe column is cooled to room temperature and the parafilm removed fromthe bottom of the column. Five column volumes of the α-MM buffer areapplied to the column and the eluate analyzed by SDS-PAGE. Typically theresulting material is about 60-95% pure, however this depends upon thecell type and the tissue-to-lysis buffer ratio used. Then the sample isapplied to a Mono Q FPLC column (Pharmacia) equilibrated with a buffercontaining 5 mM sodium phosphate, pH 7. The proteins are then elutedfrom the column with a 0-1M NaCl gradient and the gp96 fraction elutesbetween 4000 mM and 550 mM NaCl.

The procedure, however, may be modified by two additional steps, usedeither alone or in combination, to consistently produce apparentlyhomogeneous gp96-peptide complexes. One optional step involves anammonium sulfate precipitation prior to the Con A purification step andthe other optional step involves DEAE-Sepharose purification after theCon A purification step but before the Mono Q FPLC step.

In the first optional step, described by way of example as follows, thesupernatant resulting from the 100,000 g centrifugation step is broughtto a final concentration of 50% ammonium sulfate by the addition ofammonium sulfate. The ammonium sulfate is added slowly while gentlystirring the solution in a beaker placed in a tray of ice water. Thesolution is stirred from about ½ to 12 hours at 4° C. and the resultingsolution centrifuged at 6,000 rpm (Sorvall SS34 rotor) . The supernatantresulting from this step is removed, brought to 70% ammonium sulfatesaturation by the addition of ammonium sulfate solution, and centrifugedat 6,000 rpm (Sorvall SS34 rotor). The resulting pellet from this stepis harvested and suspended in PBS containing 70% ammonium sulfate inorder to rinse the pellet. This mixture is centrifuged at 6,000 rpm(Sorvall SS34 rotor) and the pellet dissolved in PBS containing 2 mMCa²⁺ and Mg²⁺. Undissolved material is removed by a brief centrifugationat 15,000 rpm (Sorvall SS34 rotor). Then, the solution is mixed with ConA Sepharose and the procedure followed as before.

In the second optional step, described by way of example as follows, thegp96 containing fractions eluted from the Con A column are pooled andthe buffer exchanged for 5 mM sodium phosphate buffer, pH 7, 300 mM NaClby dialysis, or preferably by buffer exchange on a Sephadex G25 column.After buffer exchange, the solution is mixed with DEAE-Sepharosepreviously equilibrated with 5 mM sodium phosphate buffer, pH 7, 300 mMNaCl. The protein solution and the beads are mixed gently for 1 hour andpoured into a column. Then, the column is washed with 5 mM sodiumphosphate buffer, pH 7, 300 mM NaCl, until the absorbance at 280 nmdrops to baseline. Then, the bound protein is eluted from the columnwith five volumes of 5 mM sodium phosphate buffer, pH 7, 700 mM NaCl.Protein containing fractions are pooled and diluted with 5 mM sodiumphosphate buffer, pH 7 in order to lower the salt concentration to 175mM. The resulting material then is applied to the Mono Q FPLC column(Pharmacia) equilibrated with 5 mM sodium phosphate buffer, pH 7 and theprotein that binds to the Mono Q FPLC column (Pharmacia) is eluted asdescribed before.

It is appreciated, however, that one skilled in the art may assess, byroutine experimentation, the benefit of incorporating the secondoptional step into the purification protocol. In addition, it isappreciated also that the benefit of adding each of the optional stepswill depend upon the source of the starting material.

When the gp96 fraction is isolated from the 100,000 g pellet, the pelletis suspended in 5 volumes of PBS containing either 1% sodiumdeoxycholate or 1% oxtyl glucopyranoside (but without the Mg²⁺ and Ca²⁺)and incubated on ice for 1 hour. The suspension is centrifuged at 20,000g for 30 minutes and the resulting supernatant dialyzed against severalchanges of PBS (also without the Mg²⁺ and Ca²⁺) to remove the detergent.The dialysate is centrifuged at 100,000 g for 90 minutes, thesupernatant harvested, and calcium and magnesium are added to thesupernatant to give final concentrations of 2 mM, respectively. Then thesample is purified by either the unmodified or the modified method forisolating gp96-peptide complex from the 100,000 g supernatant, seeabove.

The gp96-peptide complexes can be purified to apparent homogeneity usingthis procedure. About 10-20 g of gp96 can be isolated from 1 gcells/tissue.

Infectious Disease

In an alternative embodiment wherein it is desired to treat a patienthaving an infectious disease, the above-described methods in Sections5.2.1-5.2.3 are used to isolate hsp-peptide complexes from cellsinfected with an infectious organism, e.g., of a cell line or from apatient. Such infectious organisms include.,but are not limited to,viruses, bacteria, protozoa, fungi, and parasites as described in detailin Section 5.2.4 below.

5.2.4. Isolation of Antigenic/Immunogenic Components

It has been found that antigenic peptides and/or components can beeluted from hsp-complexes either in the presence of ATP or low pH. Theseexperimental conditions may be used to isolate peptides and/or antigeniccomponents from cells which may contain potentially useful antigenicdeterminants. Once isolated, the amino acid sequence of each antigenicpeptide may be determined using conventional amino acid sequencingmethodologies. Such antigenic molecules can then be produced by chemicalsynthesis or recombinant methods, purified, and complexed to hsps invitro.

Similarly, it has been found that potentially immunogenic peptides maybe eluted from MHC-peptide complexes using techniques well known in theart (Falk, K. et al., 1990 Nature 348:248-251; Elliott, T., et al.,1990, Nature 348:195-197; Falk, K., et al., 1991, Nature 351:290-296).

Thus, potentially immunogenic or antigenic peptides may be isolated fromeither endogenous stress protein-peptide complexes or endogenousMHC-peptide complexes for use subsequently as antigenic molecules, bycomplexing in vitro to hsps. Exemplary protocols for isolating peptidesand/or antigenic components from either of the these complexes are setforth below in Sections 5.2.4.1 and 5.2.4.2.

5.2.4.1. Peptides From Stress Protein-Peptide Complexes

Two methods may be used to elute the peptide from a stressprotein-peptide complex. One approach involves incubating the stressprotein-peptide complex in the presence of ATP. The other approachinvolves incubating the complexes in a low pH buffer.

Briefly the complex of interest is centrifuged through a Centricon 10assembly (Millipore) to remove any low molecular weight material looselyassociated with the complex. The large molecular weight fraction may beremoved and analyzed by SDS-PAGE while the low molecular weight may beanalyzed by HPLC as described below. In the ATP incubation protocol, thestress protein-peptide complex in the large molecular weight fraction isincubated with 10 mM ATP for 30 minutes at room temperature. In the lowpH protocol, acetic acid or trifluoroacetic acid (TFA) is added to thestress protein-peptide complex to give a final concentration of 10%(vol/vol) and the mixture incubated at room temperature or in a boilingwater bath or any temperature in between, for 10 minutes (See, VanBleek, et al., 1990, Nature 348:213-216; and Li, et al., 1993, EMBOJournal 12:3143-3151).

The resulting samples are centrifuged through a Centricon 10 assembly asmentioned previously. The high and low molecular weight fractions arerecovered. The remaining large molecular weight stress protein-peptidecomplexes can be reincubated with ATP or low pH to remove any remainingpeptides.

The resulting lower molecular weight fractions are pooled, concentratedby evaporation and dissolved in 0.1% TFA. The dissolved material is thenfractionated by reverse phase high pressure liquid chromatography (HPLC)using for example a VYDAC C18 reverse phase column equilibrated with0.1% TFA. The bound material is then eluted at a flow rate of about 0.8ml/min by developing the column with a linear gradient of 0 to 80%acetonitrile in 0.1% TFA. The elution of the peptides can be monitoredby OD₂₁₀ and the fractions containing the peptides collected.

5.2.4.2. Peptides from MHC-peptide Complexes

The isolation of potentially immunogenic peptides from MHC molecules iswell known in the art and so is not described in detail herein (See,Falk, et al., 1990, Nature 348:248-251; Rotzsche, at al., 1990, Nature348:252-254; Elliott, et al., 1990, Nature 348:191-197; Falk, et al.,1991, Nature 351:290-296; Demotz, et al., 1989, Nature 343:682-684;Rotzsche, et al., 1990, Science 249:283-287), the disclosures of whichare incorporated herein by reference.

Briefly, MHC-peptide complexes may be isolated by a conventionalimmunoaffinity procedure. The peptides then may be eluted from theMHC-peptide complex by incubating the complexes in the presence of about0.1% TFA in acetonitrile. The eluted peptides may be fractionated andpurified by reverse phase HPLC, as before.

The amino acid sequences of the eluted peptides may be determined eitherby manual or automated amino acid sequencing techniques well known inthe art. Once the amino acid sequence of a potentially protectivepeptide has been determined the peptide may be synthesized in anydesired amount using conventional peptide synthesis or other protocolswell known in the art.

Peptides having the same amino acid sequence as those isolated above maybe synthesized by solid-phase peptide synthesis using procedures similarto those described by Merrifield, 1963, J. Am. Chem. Soc., 85:2149.During synthesis, N-α-protected amino acids having protected side chainsare added stepwise to a growing polypeptide chain linked by itsC-terminal and to an insoluble polymeric support i.e., polystyrenebeads. The peptides are synthesized by linking an amino group of anN-α-deprotected amino acid to an α-carboxy group of an N-α-protectedamino acid that has been activated by reacting it with a reagent such asdicyclohexylcarbodiimide. The attachment of a free amino group to theactivated carboxyl leads to peptide bond formation. The most commonlyused N-α-protecting groups include Boc which is acid labile and Fmocwhich is base labile.

Briefly, the C-terminal N-α-protected amino acid is first attached tothe polystyrene beads. The N-α-protecting group is then removed. Thedeprotected α-amino group is coupled to the activated α-carboxylategroup of the next N-αprotected amino acid. The process is repeated untilthe desired peptide is synthesized. The resulting peptides are thencleaved from the insoluble polymer support and the amino acid sidechains deprotected. Longer peptides can be derived by condensation ofprotected peptide fragments. Details of appropriate chemistries, resins,protecting groups, protected amino acids and reagents are well known inthe art and so are not discussed in detail herein (See, Atherton, etal., 1989, Solid Phase Peptide Synthesis: A Practical Approach, IRLPress, and Bodanszky, 1993, Peptide Chemistry, A Practical Textbook, 2ndEd., Springer-Verlag).

Purification of the resulting peptides is accomplished usingconventional procedures, such as preparative HPLC using gel permeation,partition and/or ion exchange chromatography. The choice of appropriatematrices and buffers are well known in the art and so are not describedin detail herein.

5.2.5. Exogenous Antigenic Molecules

Antigens or antigenic portions thereof can be selected for use asantigenic molecules, for complexing to hsps, from among those known inthe art or determined by immunoassay to be able to bind to antibody orMHC molecules (antigenicity) or generate immune response(immunogenicity). To determine immunogenicity or antigenicity bydetecting binding to antibody, various immunoassays known in the art canbe used, including but not limited to competitive and non-competitiveassay systems using techniques such as radioimmunoassays, ELISA (enzymelinked immunosorbent assay), “sandwich” immunoassays, immunoradiometricassays, gel diffusion precipitin reactions, immunodiffusion assays, invivo immunoassays (using colloidal gold, enzyme or radioisotope labels,for example), western blots, immunoprecipitation reactions,agglutination assays (e.g., gel agglutination assays, hemagglutinationassays), complement fixation assays, immunofluorescence assays, proteinA assays, and immunoelectrophoresis assays, etc. In one embodiment,antibody binding is detected by detecting a label on the primaryantibody. In another embodiment, the primary antibody is detected bydetecting binding of a secondary antibody or reagent to the primaryantibody. In a further embodiment, the secondary antibody is labelled.Many means are known in the art for detecting binding in an immunoassayand are envisioned for use. In one embodiment for detectingimmunogenicity, T cell-mediated responses can be assayed by standardmethods, e.g., in vitro cytoxicity assays or in vivo delayed-typehypersensitivity assays.

Potentially useful antigens or derivatives thereof for use as antigenicmolecules can also be identified by various criteria, such as theantigen's involvement in neutralization of a pathogen's infectivity(wherein it is desired to treat or prevent infection by such a pathogen)(Norrby, 1985, Summary, in Vaccines 85, Lerner, et al. (eds.), ColdSpring Harbor Laboratory, Cold Spring Harbor, N.Y., pp. 388-389), typeor group specificity, recognition by patients' antisera or immune cells,and/or the demonstration of protective effects of antisera or immunecells specific for the antigen. In addition, where it is desired totreat or prevent a disease caused by pathogen, the antigen's encodedepitope should preferably display a small or no degree of antigenicvariation in time or amongst different isolates of the same pathogen.

Preferably, where it is desired to treat or prevent cancer, knowntumor-specific antigens or fragments or derivatives thereof are used.For example, such tumor specific or tumor-associated antigens includebut are not. limited to KS 1/4 pan-carcinoma antigen (Perez and Walker,1990, J. Immunol. 142:3662-3667; Bumal, 1988, Hybridoma 7(4):407-415);ovarian carcinoma antigen (CA125) (Yu, et al., 1991, Cancer Res. 51(2):468-475); prostatic acid phosphate (Tailer, et al., 1990, Nucl. AcidsRes. 18(16):4928); prostate specific antigen (Henttu and Vihko, 1989,Biochem. Biophys. Res. Comm. 160(2):903-910; Israeli, et al., 1993,Cancer Res. 53:227-230); melanoma-associated antigen p97 (Estin, et al.,1989, J. Natl. Cancer Inst. 81(6):445-446); melanoma antigen gp75(Vijayasardahl, et al., 1990, J. Exp. Med. 171(4):1375-1380); highmolecular weight melanoma antigen (Natali, et al., 1987, Cancer59:55-63) and prostate specific membrane antigen.

In a specific embodiment, an antigen or fragment or derivative thereofspecific to a certain tumor is selected for complexing to hsp andsubsequent administration to a patient having that tumor.

Preferably, where it is desired to treat or prevent viral diseases,molecules comprising epitopes of known viruses are used. For example,such antigenic epitopes may be prepared from viruses including, but notlimited to, hepatitis type A, hepatitis type B, hepatitis type C,influenza, varicella, adenovirus, herpes simplex type I (HSV-I), herpessimplex type II (HSV-II), rinderpest, rhinovirus, echovirus, rotavirus,respiratory syncytial virus, papilloma virus, papova virus,cytomegalovirus, echinovirus, arbovirus, huntavirus, coxsackie virus,mumps virus, measles virus, rubella virus, polio virus, humanimmunodeficiency virus type I (HIV-I), and human immunodeficiency virustype II (HIV-II).

Preferably, where it is desired to treat or prevent bacterialinfections, molecules comprising epitopes of known bacteria are used.For example, such antigenic epitopes may be prepared from bacteriaincluding, but not limited to, mycobacteria rickettsia, mycoplasma,neisseria and legionella.

Preferably, where it is desired to treat or prevent protozoalinfections, molecules comprising epitopes of known protozoa are used.For example, such antigenic epitopes may be prepared from protozoaincluding, but not limited to, leishmania, kokzidioa, and trypanosoma.

Preferably, where it is desired to treat or prevent parasiticinfections, molecules comprising epitopes of known parasites are used.For example, such antigenic epitopes may be from parasites including,but not limited to, chlamydia and rickettsia.

5.2.6. In Vitro Production of Stress Protein-Antigenic MoleculeComplexes

In an embodiment in which complexes of hsps and the eptides with whichthey are endogenously associated in vivo are not employed, complexes ofhsps to antigenic molecules are produced in vitro. As will beappreciated by those skilled in the art, the peptides either isolated bythe aforementioned procedures or chemically synthesized or recombinantlyproduced may be reconstituted with a variety of purified natural orrecombinant stress proteins in vitro to generate immunogenicnon-covalent stress protein-antigenic molecule complexes. Alternatively,exogenous antigens or antigenic/immunogenic fragments or derivativesthereof can be noncovalently complexed to stress proteins for use in theimmunotherapeutic or prophylactic vaccines of the invention. Apreferred, exemplary protocol for noncovalently complexing a stressprotein and an antigenic molecule in vitro is discussed below.

Prior to complexing, the hsps are pretreated with ATP or low pH toremove any peptides that may be associated with the hsp of interest.When the ATP procedure is used, excess ATP is removed from thepreparation by the addition of apyranase as described by Levy, et al.,1991, Cell 67:265-274. When the low pH procedure is used, the buffer isreadjusted to neutral pH by the addition of pH modifying reagents.

The antigenic molecules (1 μg) and the pretreated hsp (9 μg) are admixedto give an approximately 5 antigenic molecule: 1 stress protein molarratio. Then, the mixture is incubated for 15 minutes to 3 hours at 4° to45° C. in a suitable binding buffer such as one containing 20 mM sodiumphosphate, pH 7.2, 350 mM NaCl, 3 mM MgCl₂ and 1 mM phenyl methylsulfonyl fluoride (PMSF). The preparations are centrifuged through aCentricon 10 assembly (Millipore) to remove any unbound peptide. Theassociation of the peptides with the stress proteins can be assayed bySDS-PAGE. This is the preferred method for in vitro complexing ofpeptides isolated from MHC-peptide complexes of peptides disassociatedfrom endogenous hsp-peptide complexes.

In an alternative embodiment of the invention, preferred for producingcomplexes of hsp70 to exogenous antigenic molecules such as proteins,5-10 micrograms of purified hsp is incubated with equimolar quantitiesof the antigenic molecule in 20 mM sodium phosphate buffer pH 7.5, 0.5MNaCl, 3 mM MgCl₂ and 1 mM ADP in a volume of 100 microliter at 37° C.for 1 hr. This incubation mixture is further diluted to lml inphosphate-buffered saline.

In an alternative embodiment of the invention, preferred for producingcomplexes of gp96 or hsp90, to peptides, 5-10 micrograms of purifiedgp96 or hsp90, is incubated with equimolar or excess quantities of theantigenic peptide in a suitable buffer such as one containing 20 mMsodium phosphate buffer pH 7.5, 0.5M NaCl, 3 nM MgCl2 at 60-65° C. for5-20 min. This incubation mixture is allowed to cool to room temperatureand centrifuged one or more times if necessary, through a Centricon 10assembly (Millipore) to remove any unbound peptide.

Following complexing, the immunogenic stress protein-antigenic moleculecomplexes can optionally be assayed in vitro using for example the mixedlymphocyte target cell assay (MLTC) described below. Once immunogeniccomplexes have been isolated they can be optionally characterizedfurther in animal models using the preferred administration protocolsand excipients discussed below.

5.2.7. Determination of Immunogenicity of Stress Protein-PeptideComplexes

The purified stress protein-antigenic molecule complexes can be assayedfor immunogenicity using the MLTC assay well known in the art.

By way of example but not limitation, the following procedure can beused. Briefly, mice are injected, preferably intradermally or mucosally,with the candidate stress protein-antigenic molecule complexes. Othermice are injected with either other stress protein peptide complexes orwhole infected cells which act as positive controls for the assay. Themice are injected twice, 7-10 days apart. Ten days after the lastimmunization, the spleens are removed and the lymphocytes released. Thereleased lymphocytes may be restimulated subsequently in vitro by theaddition of dead cells that expressed the complex of interest.

For example, 8×10⁶ immune spleen cells may be stimulated with 4×10⁴mitomycin C treated or γ-irradiated (5-10,000 rads) infected cells (orcells transfected with an appropriate gene, as the case may be) in 3 mlRPMI medium containing 10% fetal calf serum. In certain cases 33%secondary mixed lymphocyte culture supernatant may be included in theculture medium as a source of T cell growth factors (See, Glasebrook, etal., 1980, J. Exp. Med. 151:876). To test the primary cytotoxic T cellresponse after immunization, spleen cells may be cultured withoutstimulation. In some experiments spleen cells of the immunized mice mayalso be restimulated with antigenically distinct cells, to determine thespecificity of the cytotoxic T cell response.

Six days later the cultures are tested for cytotoxicity in a 4 hour⁵¹Cr-release assay (See, Palladino, et al., 1987, Cancer Res.47:5074-5079 and Blachere, at al., 1993, J. Immunotherapy 14:352-356).In this assay, the mixed lymphocyte culture is added to a target cellsuspension to give different effector:target (E:T) ratios (usually 1:1to 40:1). The target cells are prelabelled by incubating 1×10⁶targetcells in culture medium containing 20 mCi ⁵¹Cr/ml for one hour at 37° C.The cells are washed three times following labeling. Each assay point(E:T ratio) is performed in triplicate and the appropriate controlsincorporated to measure spontaneous ⁵¹Cr release (no lymphocytes addedto assay) and 100% release (cells lysed with detergent). Afterincubating the cell mixtures for 4 hours, the cells are pelletted bycentrifugation at 200 g for 5 minutes. The amount of ⁵¹Cr released intothe supernatant is measured by a gamma counter. The percent cytotoxicityis measured as cpm in the test sample minus spontaneously released cpmdivided by the total detergent released cpm minus spontaneously releasedcpm.

In order to block the MHC class I cascade a concentrated hybridomasupernatant derived from K-44 hybridoma cells (an anti-MHC class Ihybridoma) is added to the test samples to a final concentration of12.5%.

5.3. Combination With Adoptive Immunotherapy

Adoptive immunotherapy refers to a therapeutic approach for treatingcancer or infectious diseases in which immune cells are administered toa host with the aim that the cells mediate either directly or indirectlyspecific immunity to tumor cells and/or antigenic components orregression of the tumor or treatment of infectious diseases, as the casemay be. (See U.S. patent application Ser. No. 08/527,546, filed Sep. 13,1995, which is incorporated by reference herein in its entirety.) As anoptional step, in accordance with the methods described herein, APC aresensitized with hsps noncovalently complexed with antigenic (orimmunogenic) molecules and used in adoptive immunotherapy.

In a specific embodiment, therapy by administration of hsp-peptidecomplexes, using any desired route of administration, may optionally becombined with adoptive immunotherapy using APC sensitized withhsp-antigenic molecule complexes. As described in Section 5 herein, thehsp-peptide complex-sensitized APC can be administered alone, incombination with hsp-peptide complexes, or before or afteradministration of hsp-peptide complexes. Furthermore, the mode ofadministration can be varied, including but not limited to, e.g.,subcutaneously, intravenously or intramuscularly, although intradermallyor mucosally is preferred.

5.3.1. Obtaining Macrophages and Antigen-Presenting Cells

The antigen-presenting cells, including but not limited to macrophages,dendritic cells and B-cells, are preferably obtained by production invitro from stem and progenitor cells from human peripheral blood or bonemarrow as described by Inaba, K., et al., 1992, J. Exp. Med.176:1693-1702.

APC can be obtained by any of various methods known in the art. In apreferred aspect human macrophages are used, obtained from human bloodcells. By way of example but not limitation, macrophages can be obtainedas follows:

Mononuclear cells are isolated from peripheral blood of a patient(preferably the patient to be treated), by Ficoll-Hypaque gradientcentrifugation and are seeded on tissue culture dishes which arepre-coated with the patient's own serum or with other AB+human serum.The cells are incubated at 37° C. for 1 hour, then non-adherent cellsare removed by pipetting. To the adherent cells left in the dish, isadded cold (4° C.) 1 mM EDTA in phosphate-buffered saline and the dishesare left at room temperature for 15 minutes. The cells are harvested,washed with RPMI buffer and suspended in RPMI buffer. Increased numbersof macrophages may be. obtained by incubating at 37° C. withmacrophage-colony stimulating factor (M-CSF); increased numbers ofdendritic cells may be obtained by incubating withgranulocyte-macrophage-colony stimulating factor (GM-CSF) as describedin detail by Inaba, K., et al., 1992, J. Exp. Med. 176:1693-1702.

5.3.2. Sensitization of Macrophages and Antigen Presenting. Cells WithHsp-Peptide Complexes

APC are sensitized with hsp noncovalently bound to antigenic moleculespreferably by incubating the cells in vitro with the complexes. The APCare sensitized with complexes of hsps and antigenic molecules byincubating in vitro with the hsp-complex at 37° C. for 15 minutes to 24hours. By way of example but not limitation, 4-10⁷ macrophages can beincubated with 10 microgram gp96-peptide complexes per ml or 100microgram hsp90-peptide complexes per ml at 37° C. for 15 minutes-24hours in 1 ml plain RPMI medium. The cells are washed three times andresuspended in a physiological medium preferably sterile, at aconvenient concentration (e.g., 1×10⁷/ml) for injection in a patient.Preferably, the patient into which the sensitized APCs are injected isthe patient from which the APC were originally isolated (autologousembodiment).

Optionally, the ability of sensitized APC to stimulate, for example, theantigen-specific, class I-restricted cytotoxic T-lymphocytes (CTL) canbe monitored by their ability to stimulate CTLs to release tumornecrosis factor, and by their ability to act as targets of such CTLs.

5.3.3. Reinfusion of Sensitized APC

The hsp-antigenic molecule-sensitized APC are reinfused into the patientsystemically, preferably intravenously, by conventional clinicalprocedures. These activated cells are reinfused, preferentially bysystemic administration into the autologous patient. Patients generallyreceive from about 10⁶ to about 10¹² sensitized macrophages, dependingon the condition of the patient. In some regimens, patients mayoptionally receive in addition a suitable dosage of a biologicalresponse modifier including but not limited to the cytokines IFN-α,IFN-γ, IL-2, IL-4, IL-6, TNF or other cytokine growth factor.

5.4. Formulation, Administration & Kits

Hsp-antigenic molecule complexes of the invention may be formulated intopharmaceutical preparations for administration to mammals, preferablyhumans, for treatment or prevention of cancer or infectious diseases.Compositions comprising a compound of the invention formulated in acompatible pharmaceutical carrier may be prepared, packaged, andlabelled for treatment of the indicated tumor(s), such as human sarcomasand carcinomas, e.g., fibrosarcoma, myxosarcoma, liposarcoma,chondrosarcoma, osteogenic sarcoma, chordoma, angiosarcoma,endotheliosarcoma, lymphangiosarcoma, lymphangioendotheliosarcoma,synovioma, mesothelioma, Ewing's tumor, leiomyosarcoma,rhabdomyosarcoma, colon carcinoma, pancreatic cancer, breast cancer,ovarian cancer, prostate cancer, squamous cell carcinoma, basal cellcarcinoma, adenocarcinoma, sweat gland carcinoma, sebaceous glandcarcinoma, papillary carcinoma, papillary adenocarcinomas,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma,seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testiculartumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma,epithelial carcinoma, glioma, astrocytoma, medulloblastoma,craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acousticneuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma,retinoblastoma; leukemias, e.g., acute lymphocytic leukemia and acutemyelocytic leukemia (myeloblastic, promyelocytic, myelomonocytic,monocytic and erythroleukemia); chronic leukemia (chronic myelocytic(granulocytic) leukemia and chronic lymphocytic leukemia); andpolycythemia vera, lymphoma (Hodgkin's disease and non-Hodgkin'sdisease), multiple myeloma, Waldenström's macroglobulinemia, and heavychain disease. Alternatively, it can be labeled for treatment of theappropriate infectious disease. Alternatively, pharmaceuticalcompositions may be formulated for treatment of appropriate infectiousdiseases.

Drug solubility and the site of absorption are factors which should beconsidered when choosing the route of administration of a therapeuticagent. In an embodiment of the invention, hsp-antigenic moleculecomplexes may be administered using any desired route of administration,and preferably intradermally or mucosally. Advantages of intradermal ormucosal administration include use of lower doses and rapid absorption,respectively. Mucosal routes of administration include, but are notlimited to, oral, rectal and nasal administration. Preparations formucosal administrations are suitable in various formulations asdescribed below.

If the complex is water-soluble, then it may be formulated in anappropriate buffer, for example, phosphate buffered saline or otherphysiologically compatible solutions, preferably sterile. Alternatively,if the resulting complex has poor solubility in aqueous solvents, thenit may be formulated with a non-ionic surfactant such as Tween, orpolyethylene glycol. Thus, the compounds and their physiologicallyacceptable solvates may be formulated for administration by inhalationor insufflation (either through the mouth or the nose) or oral, buccal,parenteral, or rectal administration or, in the case of tumors, directlyinjected into a solid tumor.

For oral administration, the pharmaceutical preparation may be in liquidform, for example, solutions, syrups or suspensions, or may be presentedas a drug product for reconstitution with water or other suitablevehicle before use. Such liquid preparations may be prepared byconventional means with pharmaceutically acceptable additives such assuspending agents (e.g., sorbitol syrup, cellulose derivatives orhydrogenated edible fats); emulsifying agents (e.g., lecithin oracacia); non-aqueous vehicles (e.g., almond oil, oily esters, orfractionated vegetable oils); and preservatives (e.g., methyl orpropyl-p-hydroxybenzoates or sorbic acid). The pharmaceuticalcompositions may take the form of, for example, tablets or capsulesprepared by conventional means with pharmaceutically acceptableexcipients such as binding agents (e.g., pregelatinized maize starch,polyvinyl-pyrrolidone or hydroxypropyl methylcellulose); fillers (e.g.,lactose, microcrystalline cellulose or calcium hydrogen phosphate);lubricants (e.g., magnesium stearate, talc or silica); disintegrants(e.g., potato starch or sodium starch glycolate); or wetting agents(e.g., sodium lauryl sulphate). The tablets may be coated by methodswell-known in the art.

Preparations for oral administration may be suitably formulated to givecontrolled release of the active compound.

For buccal administration, the compositions may take the form of tabletsor lozenges formulated in conventional manner.

The compounds may be formulated for parenteral administration byinjection, e.g., by bolus injection or continuous infusion. Formulationsfor injection may be presented in unit dosage form, e.g., in ampoules orin multi-dose containers, with an added preservative. The compositionsmay take such forms as suspensions, solutions or emulsions in oily oraqueous vehicles, and may contain formulatory agents such as suspending,stabilizing and/or dispersing agents. Alternatively, the activeingredient may be in powder form for constitution with a suitablevehicle, e.g., sterile pyrogen-free water, before use.

The compounds may also be formulated in rectal compositions such assuppositories or retention enemas, e.g., containing conventionalsuppository bases such as cocoa butter or other glycerides.

In addition to the formulations described previously, the compounds mayalso be formulated as a depot preparation. Such long acting formulationsmay be administered by implantation (for example, subcutaneously orintramuscularly) or by intramuscular injection. Thus, for example, thecompounds may be formulated with suitable polymeric or hydrophobicmaterials (for example, as an emulsion in an acceptable oil) or ionexchange resins, or as sparingly soluble derivatives, for example, as asparingly soluble salt. Liposomes and emulsions are well known examplesof delivery vehicles or carriers for hydrophilic drugs.

For administration by inhalation, the compounds for use according to thepresent invention are conveniently delivered in the form of an aerosolspray presentation from pressurized packs or a nebulizer, with the useof a suitable propellant, e.g., dichlorodifluoromethane,trichlorofluoromethane, dichlorotetrafluoroethane, carbon dioxide orother suitable gas. In the case of a pressurized aerosol the dosage unitmay be determined by providing a valve to deliver a metered amount.Capsules and cartridges of, e.g., gelatin for use in an inhaler orinsufflator may be formulated containing a powder mix of the compoundand a suitable powder base such as lactose or starch.

The compositions may, if desired, be presented in a pack or dispenserdevice which may contain one or more unit dosage forms containing theactive ingredient. The pack may for example comprise metal or plasticfoil, such as a blister pack. The pack or dispenser device may beaccompanied by instructions for administration.

The invention also provides kits for carrying out the therapeuticregimens of the invention. Such kits comprise in one or more containerstherapeutically or prophylactically effective amounts of thehsp-antigenic molecule complexes, preferably purified, inpharmaceutically acceptable form. The kits optionally further comprisein a second container the sensitized APC of the invention, preferablypurified. The hsp-antigenic molecule complex in a vial of a kit of theinvention may be in the form of a pharmaceutically acceptable solution,e.g., in combination with sterile saline, dextrose solution, or bufferedsolution, or other pharmaceutically acceptable sterile fluid.Alternatively, the complex may be lyophilized or desiccated; in thisinstance, the kit optionally further comprises in a container apharmaceutically acceptable solution (e.g., saline, dextrose solution,etc.), preferably sterile, to reconstitute the complex to form asolution for injection purposes.

In another embodiment, a kit of the invention further comprises a needleor syringe, preferably packaged in sterile form, for injecting thecomplex, and/or a packaged alcohol pad. Instructions are optionallyincluded for administration of hsp-antigenic molecule complexes by aclinician or by the patient.

5.5. Target Infectious Diseases

Infectious diseases that can be treated or prevented by the methods ofthe present invention are caused by infectious agents including, but notlimited to, viruses, bacteria, fungi protozoa and parasites.

Viral diseases that can be treated or prevented by the methods of thepresent invention include, but are not limited to, those caused byhepatitis type A, hepatitis type B, hepatitis type C, influenza,varicella, adenovirus, herpes simplex type I (HSV-I), herpes simplextype II (HSV-II), rinderpest, rhinovirus, echovirus, rotavirus,respiratory syncytial virus, papilloma virus, papova virus,cytomegalovirus, echinovirus, arbovirus, huntavirus, coxsackie virus,mumps virus, measles virus, rubella virus, polio virus, humanimmunodeficiency virus type I (HIV-I), and human immunodeficiency virustype II (HIV-II).

Bacterial diseases that can be treated or prevented by the methods ofthe present invention are caused by bacteria including, but not limitedto, mycobacteria rickettsia, mycoplasma, neisseria and legionella.

Protozoal diseases that can be treated or prevented by the methods ofthe present invention are caused by protozoa including, but not limitedto, leishmania, kokzidioa, and trypanosoma.

Parasitic diseases that can be treated or prevented by the methods ofthe present invention are caused by parasites including, but not limitedto, chlamydia and rickettsia.

5.6. Target Cancers

Cancers that can be treated or prevented by the methods of the presentinvention include, but are not limited to human sarcomas and carcinomas,e.g., fibrosarcoma, myxosarcoma, liposarcoma, chondrosarcoma, osteogenicsarcoma, chordoma, angiosarcoma, endotheliosarcoma, lymphangiosarcoma,lymphangioendotheliosarcoma, synovioma, mesothelioma, Ewing's tumor,leiomyosarcoma, rhabdomyosarcoma, colon carcinoma, pancreatic cancer,breast cancer, ovarian cancer, prostate cancer, squamous cell carcinoma,basal cell carcinoma, adenocarcinoma, sweat gland carcinoma, sebaceousgland carcinoma, papillary carcinoma, papillary adenocarcinomas,cystadenocarcinoma, medullary carcinoma, bronchogenic carcinoma, renalcell carcinoma, hepatoma, bile duct carcinoma, choriocarcinoma,seminoma, embryonal carcinoma, Wilms' tumor, cervical cancer, testiculartumor, lung carcinoma, small cell lung carcinoma, bladder carcinoma,epithelial carcinoma, glioma, astrocytoma, medulloblastoma,craniopharyngioma, ependymoma, pinealoma, hemangioblastoma, acousticneuroma, oligodendroglioma, meningioma, melanoma, neuroblastoma,retinoblastoma; leukemias, e.g., acute lymphocytic leukemia and acutemyelocytic leukemia (myeloblastic, promyelocytic, myelomonocytic,monocytic and erythroleukemia) ; chronic leukemia (chronic myelocytic(granulocytic) leukemia and chronic lymphocytic leukemia); andpolycythemia vera, lymphoma (Hodgkin's disease and non-Hodgkin'sdisease), multiple myeloma, Waldenström's macroglobulinemia, and heavychain disease. Specific examples of such cancers are described in thesections below.

In a specific embodiment the cancer is metastatic. In another specificembodiment, the patient having a cancer is immunosuppressed by reason ofhaving undergone anti-cancer therapy (e.g., chemotherapy radiation)prior to administration of the hsp-antigenic molecule complexes oradministration of the hsp-sensitized APC.

5.6.1. Colorectal Cancer Metastatic to the Liver

In 1992, approximately 150,000 Americans were diagnosed with colorectalcancer and more than 60,000 died as a result of colorectal metastases.At the time of their deaths, 80 percent of patients with colorectalcancer have metastatic disease involving the liver, and one-half ofthese patients have no evidence of other (extrahepatic) metastases. Mostmetastatic tumors of the liver are from gastrointestinal primaries.Unfortunately, the natural history of metastatic liver lesions carries agrave prognosis and systemic chemotherapy regimens have been unable toinduce significant response rates or alter length of survival (Drebin,J. A., et al., in Current Therapy In Oncology, ed. J. E. Niederhuber, B.C. Decker, Mosby, 1993, p.426).

Colorectal cancer initially spreads to regional lymph nodes and thenthrough the portal venous circulation to the liver, which represents themost common visceral site of metastasis. The symptoms that lead patientswith colorectal cancer to seek medical care vary with the anatomicallocation of the lesion. For example, lesions in the ascending colonfrequently ulcerate, which leads to chronic blood loss in the stool.

Radical resection offers the greatest potential for cure in patientswith invasive colorectal cancer. Before surgery, the CEA titer isdetermined. Radiation therapy and chemotherapy are used in patients withadvanced colorectal cancer. Results with chemotherapeutic agents (e.g.,5-fluorouracil) are mixed and fewer than 25 percent of patientsexperience a greater than 50 percent reduction in tumor mass (Richards,2d., F., et al., 1986, J. Clin. Oncol. 4:565).

Patients with widespread metastases have limited survival and systemicchemotherapy has little impact in this group of patients. In addition,systemically administered chemotherapy is often limited by the severityof toxicities associated with the various agents, such as severediarrhea, mucositis and/or myelosuppression. Other techniques, includinghepatic radiation, systemic chemotherapy, hepatic arterial ligation,tumor embolization and immunotherapy have all been explored, but, forthe most part, have proven ineffectual in prolonging patient survival.

In a specific embodiment, the present invention provides compositionsand methods for enhancing tumor specific immunity in individualssuffering from colorectal cancer metastasized to the liver, in order toinhibit the progression of the neoplastic disease. Preferred methods oftreating these neoplastic diseases comprise administering a compositionof autologous hsp noncovalently bound to peptide complexes, whichelicits tumor-specific immunity against the tumor cells. Mostspecifically, the use of a composition of the invention, comprisinggp96, can result in nearly complete inhibition of liver cancer growth incancer patients, without inducing toxicity and thus providing a dramatictherapeutic effect.

Accordingly, as an example of the method of the invention,gp96-antigenic molecule complexes are administered to a patientdiagnosed with colorectal cancer, with or without liver metastasis, viaone of many different routes of administration, the preferred routebeing intradermally at different anatomical sites, e.g., left arm, rightarm, left belly, right belly, left thigh, right thigh, etc. The site ofinjection is varied for each weekly injection as described in Sections 7and 8. Exemplary primary and metastatic cancers that can be prevented ortreated according to the methods of the invention are described indetail in the sections which follow and by way of example, infra.

5.6.2. Hepatocellular Carcinoma

Hepatocellular carcinoma is generally a disease of the elderly in theUnited States. Although many factors may lead to hepatocellularcarcinoma, the disease is usually limited to those persons withpreexisting liver disease. Approximately 60 to 80 percent of patients inthe United States with hepatocellular carcinoma have a cirrhotic liverand about four percent of individuals with a cirrhotic liver eventuallydevelop hepatocellular carcinoma (Niederhuber, J. E., (ed.), 1993,Current Therapy in Oncology, B. C. Decker, Mosby). The risk is highestin patients whose liver disease is caused by inherited hemochromatosisor hepatic B viral infection (Bradbear, R. A., et al., 1985, J. Natl.Cancer Inst. 75:81; Beasley, R. P., et al., 1981, Lancet 2:1129). Othercauses of cirrhosis that can lead to hepatocellular carcinoma includealcohol abuse and hepatic fibrosis caused by chronic administration ofmethotrexate. The most frequent symptoms of hepatocellular carcinoma arethe development of a painful mass in the right upper quadrant orepigastrium, accompanied by weight loss. In patients with cirrhosis, thedevelopment of hepatocellular carcinoma is preceded by ascites, portalhypertension and relatively abrupt clinical deterioration. In mostcases, abnormal values in standard liver function tests such as serumaminotransferase and alkaline phosphatase are observed.

CT scans of the liver are used to determine the anatomic distribution ofhepatocellular carcinoma and also provide orientation for percutaneousneedle biopsy. Approximately 70 percent of patients with hepatocellularcarcinoma have an elevated serum alpha-fetoprotein concentration(McIntire, K. R., et al., 1975, Cancer Res. 35:991) and itsconcentration correlates with the extent of the disease.

Radical resection offers the only hope for cure in patients withhepatocellular carcinoma. Such operative procedures are associated withfive-year survival rates of 12 to 30 percent. Liver transplantation mayimprove survival of some younger individuals. However, most patients arenot surgical candidates because of extensive cirrhosis multifocal tumorpattern or scarcity of compatible donor organs. Chemotherapeutic agentshave been administered either by intravenous route or through anintrahepatic arterial catheter. Such therapy has sometimes been combinedwith irradiation to the liver. Reductions in the size of measurabletumors of 50% or more have been reported in some patients treated witheither systemic doxorubicin or 5-fluorouracil. However, chemotherapyoften induces immunosuppression and rarely causes the tumor to disappearcompletely and the duration of response is short. The prognosis forpatients with hepatocellular carcinoma is negatively correlated withcirrhosis and metastases to the lungs or bone. Median survival forpatients is only four to six months. In another specific embodiment, thepresent invention provides compositions and methods for enhancingspecific immunity in individuals suffering from hepatocellular carcinomain order to inhibit the progression of the neoplastic disease andultimately irradiate all preneoplastic and neoplastic cells.

5.6.3. Breast Cancer

Another specific aspect of the invention relates to the treatment ofbreast cancer. The American Cancer Society estimated that in 1992180,000 American women were diagnosed with breast cancer and 46,000succumbed to the disease (Niederhuber, J. E. ed. Current Therapy inOncology B. C. Decker, Mosby, 1993). This makes breast cancer the secondmajor cause of cancer death in women, ranking just behind lung cancer. Adisturbing fact is the observation that breast cancer has beenincreasing at a rate of 3 percent per year since 1980 (Niederhuber, J.E., ed. Current Therapy in Oncology, B.C. Decker, Mosby, (1993)). Thetreatment of breast cancer presently involves surgery, radiation,hormonal therapy and/or chemotherapy. Consideration of two breast cancercharacteristics, hormone receptors and disease extent, has governed howhormonal therapies and standard-dose chemotherapy are sequenced toimprove survival and maintain or improve quality of life. A wide rangeof multidrug regimens have been used as adjuvant therapy in breastcancer patients, including, but not limited to combinations of 2cyclophosphamide, doxorubicin, vincristine methotrexate, 5-fluorouraciland/or leucovorin. In a specific embodiment, the present inventionprovides hsp compositions and methods for enhancing specific immunity topreneoplastic and neoplastic mammary cells in women. The presentinvention also provides compositions and methods for preventing thedevelopment of neoplastic cells in women at enhanced risk for breastcancer, and for inhibiting cancer cell proliferation and metastasis.These compositions can be applied alone or in combination with eachother or with biological response modifiers.

10 5.7. Autologous Embodiment

The specific immunogenicity of hsps derives not from hsps per se, butfrom the peptides bound to them. In a preferred embodiment of theinvention directed to the use of autologous complexes of hsp-peptides ascancer vaccines, two of the most intractable hurdles to cancerimmunotherapy are circumvented. First is the possibility that humancancers, like cancers of experimental animals, are antigenicallydistinct. In an embodiment of the present invention, hsps chaperoneantigenic peptides of the cancer cells from which they are derived andcircumvent this hurdle. Second, most current approaches to cancerimmunotherapy focus on determining the CTL-recognized epitopes of cancercell lines. This approach requires the availability of cell lines andCTLs against cancers. These reagents are unavailable for an overwhelmingproportion of human cancers. In an embodiment of the present inventiondirected to the use of autologous complexes of hsp-peptides, cancerimmunotherapy does not depend on the availability of cell lines or CTLsnor does it require definition of the antigenic epitopes of cancercells. These advantages make autologous hsps noncovalently bound topeptide complexes attractive immunogens against cancer.

5.8. Prevention and Treatment of Primary and Metastatic NeoplasticDiseases

There are many reasons why immunotherapy as provided by the presentinvention is desired for use in cancer patients. First, if cancerpatients are immunosuppressed, surgery with anesthesia and subsequentchemotherapy may worsen the immunosuppression. With appropriateimmunotherapy in the preoperative period, this immunosuppression may beprevented or reversed. This could lead to fewer infectious complicationsand to accelerated wound healing. Second, tumor bulk is minimalfollowing surgery and immunotherapy is most likely to be effective inthis situation. A third reason is the possibility that tumor cells areshed into the circulation at surgery and effective immunotherapy appliedat this time can eliminate these cells.

The preventive and therapeutic methods of the invention are directed atenhancing the immunocompetence of the cancer patient either beforesurgery, at or after surgery, and to induce tumor-specific immunity tocancer cells, with the objective being inhibition of cancer, and withthe ultimate clinical objective being total cancer regression anderadication.

5.9. Monitoring of Effects During Cancer Prevention and Immunotherapywith Hsp-peptide Complexes

The effect of immunotherapy with hsp-antigenic molecule complexes ondevelopment and progression of neoplastic diseases can be monitored byany methods known to one skilled in the art, including but not limitedto measuring: a) delayed hypersensitivity as an assessment of cellularimmunity; b) activity of cytolytic T-lymphocytes in vitro; c) levels oftumor specific antigens, e.g., carcinoembryonic (CEA) antigens; d)changes in the morphology of tumors using techniques such as a computedtomographic (CT) scan; and e) changes in levels of putative biomarkersof risk for a particular cancer in individuals at high risk, and f)changes in the morphology of tumors using a sonogram.

5.9.1. Delayed Hypersensitivity Skin Test

Delayed hypersensitivity skin tests are of great value in the overallimmunocompetence and cellular immunity to an antigen. Inability to reactto a battery of common skin antigens is termed anergy (Sato, T., et al.,1995, Clin. Immunol. Pathol. 74:35-43).

Proper technique of skin testing requires that the antigens be storedsterile at 4° C., protected from light and reconstituted shorted beforeuse. A 25- or 27-gauge needle ensures intradermal, rather thansubcutaneous, administration of antigen. Twenty-four and 48 hours afterintradermal administration of the antigen, the largest dimensions ofboth erythema and induration are measured with a ruler. Hypoactivity toany given antigen or group of antigens is confirmed by testing withhigher concentrations of antigen or, in ambiguous circumstances, by arepeat test with an intermediate test.

5.9.2. Activity of Cytolytic T-lymphocytes In Vitro

8×10⁶ peripheral blood derived T lymphocytes isolated by theFicoll-Hypaque centrifugation gradient technique, are restimulated with4×10⁴ mitomycin C treated tumor cells in 3ml RPMI medium containing 10%fetal calf serum. In some experiments, 33% secondary mixed lymphocyteculture supernatant or IL-2, is included in the culture medium as asource of T cell growth factors.

In order to measure the primary response of cytolytic T-lymphocytesafter immunization, T cells are cultured without the stimulator tumorcells. In other experiments, T cells are restimulated with antigenicallydistinct cells. After six days, the cultures are tested for cytotoxicityin a 4 hour ⁵¹Cr-release assay. The spontaneous ⁵¹Cr-release of thetargets should reach a level less than 20%. For the anti-MHC class Iblocking activity, a tenfold concentrated supernatant of W6/32 hybridomais added to the test at a final concentration of 12.5% (Heike M., etal., J. Immunotherapy 15:165-174).

5.9.3. Levels of Tumor Specific Antigens

Although it may not be possible to detect unique tumor antigens on alltumors, many tumors display antigens that distinguish them from normalcells. The monoclonal antibody reagents have permitted the isolation andbiochemical characterization of the antigens and have been invaluablediagnostically for distinction of transformed from nontransformed cellsand for definition of the cell lineage of transformed cells. Thebest-characterized human tumor-associated antigens are the oncofetalantigens. These antigens are expressed during embryogenesis, but areabsent or very difficult to detect in normal adult tissue. The prototypeantigen is carcinoembryonic antigen (CEA), a glycoprotein found on fetalgut and human colon cancer cells, but not on normal adult colon cells.Since CEA is shed from colon carcinoma cells and found in the serum, itwas originally thought that the presence of this antigen in the serumcould be used to screen patients for colon cancer. However, patientswith other tumors, such as pancreatic and breast cancer, also haveelevated serum levels of CEA. Therefore, monitoring the fall and rise ofCEA levels in cancer patients undergoing therapy has proven useful forpredicting tumor progression and responses to treatment.

Several other oncofetal antigens have been useful for diagnosing andmonitoring human tumors, e.g., alpha-fetoprotein, an alpha-globulinnormally secreted by fetal liver and yolk sac cells, is found in theserum of patients with liver and germinal cell tumors and can be used asa marker of disease status.

5.9.4. Computed Tomographic (CT) Scan

CT remains the choice of techniques for the accurate staging of cancers.CT has proved more sensitive and specific than any other imagingtechniques for the detection of metastases.

5.9.5. Measurement of Putative Biomarkers

The levels of a putative biomarker for risk of a specific cancer aremeasured to monitor the effect of hsp noncovalently bound to peptidecomplexes. For example, in individuals at enhanced risk for prostatecancer, serum prostate-specific antigen (PSA) is measured by theprocedure described by Brawer, M. K., et al., 1992, J. Urol.147:841-845, and Catalona, W. J., et al., 1993, JAMA 270:948-958; or inindividuals at risk for colorectal cancer, CEA is measured as describedabove in Section 4.5.3; and in individuals at enhanced risk for breastcancer, 16-α-hydroxylation of estradiol is measured by the proceduredescribed by Schneider, J. et al., 1982, Proc. Natl. Acad. Sci. ISA79:3047-3051.

5.9.6. Sonogram

A sonogram remains an alternative choice of technique for the accuratestaging of cancers.

6. EXAMPLE Methylcholamtherne (Meth A) -Induced Sarcoma Model

Gp96-antigenic molecule complexes, administered intradermally in lowdoses, can prevent development of cancer and can mediate therapy ofpre-existing cancers.

6.1. Prevention Modality

(a) Materials and Methods

Gp96-antigenic molecule complexes were derived from Meth A sarcoma cellsas described in Section 5.2.3.

Five groups of BALB/cJ mice (from The Jackson Laboratories, Bar Harbor,Maine) were given the following treatments: A) Intradermal injection ofbuffer solution; B) Intradermal injection of 1 microgram gp96-antigenicmolecule complexes derived from Meth A sarcoma cells; and C) Intradermalinjection of 5 microgram gp96-antigenic molecule complexes derived fromMeth A sarcoma cells.

The above treatments were administered twice, at different sites, atweekly intervals before injecting intradermally, 1 week after the secondinjection 1×10⁵ Meth A sarcoma cells. Tumor growth was monitored bymeasuring the average tumor diameter.

(b) Results

Tumor growth was comparable in groups A and C, i.e., mice receiving thecontrol buffer solution or the 5 microgram dose of gp95-peptidecomplexes derived from Meth A sarcoma cells. In mice treated with 1microgram gp96-peptide complexes (B), tumor growth was markedlyinhibited compared with the mice receiving the buffer control or the 5microgram gp96-antigenic molecule complex (FIG. 1A-C). The mostpreferred dose of gp96-antigenic molecule complex per administration was0.5 to 2.0 micrograms (data not shown).

Thus, intradermal administration of low doses of antigenic moleculecomplexes, described herein, represents an approach to prevention ofcancer with potential applicability to a wide range of cancers,infectious diseases or immunological disorders.

7. EXAMPLES Adoptive Transfer of sensitized Macrophages, Alone or inCombination with Administration of HSP-Peptide Complexes

Autologous human macrophages are sensitized with autologous human gp96noncovalently bound to an antigenic/immunogenic molecule. The sensitizedmacrophages are administered to the human patient at approximately thesame time as, or before, or after the administration of thegp96-antigenic molecule complex.

7.1. Materials and Methods

Macrophages are obtained as follows: mononuclear cells are isolated fromperipheral blood of the human patient to be treated, by Ficoll-Hypaquegradient centrifugation and are seeded on tissue culture dishes whichare pre-coated with the patient's own serum or with other AB+humanserum. The cells are incubated at 37° C. for 1 hour, then non-adherentcells are removed by pipetting. To the adherent cells left in the dish,is added cold (40° C.) 1 mM EDTA in phosphate-buffered saline and thedishes are left at room temperature for 15 minutes. The cells areharvested, washed with RPMI buffer and suspended in RPMI buffer.Increased numbers of macrophages may be obtained by incubating at 37° C.with macrophage-colony stimulating factor (M-CSF); increased numbers ofdendritic cells may be obtained by incubating withgranulocyte-macrophage-colony stimulating factor (GM-CSF) as describedin detail by Inaba, K., et al., 1992, J. Exp. Med. 176:1693-1702.

The macrophages (4×10⁷) are then incubated at 37° C. for 3 hour in 1 mlRPMI containing 50 μg gp96-peptide complexes derived from the autologoustumor or from autologous liver, using methods as described in Section5.2.3. The macrophages are then washed 3 times and resuspended at aconcentrate of 1×10⁷/ml in RPMI medium. 200 microliters of thissuspension is administered as described in the experimental protocolbelow.

7.2. TREATMENT OF HEPATOCELLULAR CARCINOMA

Five groups of human patients with hepatocellular carcinoma are injectedwith autologous macrophages sensitized with hsp-peptide complexesderived from their own tumors post surgery. Treatment with hsp-peptidecomplexes is started any time after surgery. However, if the patient hasreceived chemotherapy, sensitized macrophages alone or in combinationwith an hsp-peptide complexes are usually administered after an intervalof four weeks or more so as to allow the immune system to recover. Theimmunocompetence of the patient is tested by procedures described insections 5.7 above.

The preferred therapeutic regimen includes weekly injections of thesensitized macrophages in combination with an hsp-peptide complexdissolved in saline or other physiologically compatible solution.Sensitized macrophages may be administered at approximately the sametime with an hsp-peptide complex or one may be administered prior toadministration of the other.

The dosage used for hsp70 or gp96 is in the range of 0.1 to 9micrograms, with the preferred dosage being 0.5-2.0 micrograms. Thedosage used for hsp90, is in the range of 5 to 500 micrograms, with thepreferred dosage being about 10 micrograms.

The site of injection is varied each time, for example, the firstinjection is given intradermally on the left arm, the second injectionon the right arm, the third injection on the left abdominal region, thefourth injection on the right abdominal region, the fifth injection onthe left thigh, the sixth injection on the right thigh, etc. The samesite is repeated after a gap of one or more injections. In addition,injections are split and each half of the dose is administered at adifferent site on the same day.

Overall, the first four to six injections are given at weekly intervals.Subsequently, two injections are given at two-week intervals; followedby a regimen of injections at monthly intervals. The effect of therapyis monitored by measuring: a) delayed hypersensitivity as an assessmentof cellular immunity; b) activity of cytolytic T-lymphocytes in vitro;c) levels of tumor specific antigens, e.g., carcinoembryonic (CEA)antigens; d) changes in the morphology of tumors using techniques suchas a computed tomographic (CT) scan; and e) changes in putativebiomarkers of risk for a particular cancer in individuals at high risk.

Depending on the results obtained, as described above in Section 5.10,the therapeutic regimen may be modified to maintain and/or boost theimmunological responses of the patient, with the ultimate goal ofachieving tumor regression and complete eradication of cancer cells.

8. EXAMPLE Administration of HSP-Peptide complexes in the Treatment ofColorectal Cancer

Hsp-peptide complexes (gp96, hsp70, , hsp90, or a combination thereof)are administered as adjuvant therapy and as prophylactic adjuvanttherapy in patients after complete reduction of colorectal cancer toeliminate undetectable micrometastases and to improve survival.

The therapeutic and prophylactic regimens used in patients sufferingfrom colorectal cancer are the same as those described in Section 7above for patients recovering with hepatocellular carcinoma. The methodsof monitoring of patients under clinical evaluation for prevention andtreatment of colorectal cancer is done by procedures described inSection 5.7. Specifically, CEA levels are measured as a useful monitorof tumor regression and/or recurrence (Mayer, R. J., et al., 1978,Cancer 42:1428).

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of theinvention in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and accompanyingfigures. Such modifications are intended to fall within the scope of theappended claims.

Various publications are cited herein, the disclosures of which areincorporated by reference in their entireties.

What is claimed is:
 1. A method of inhibiting growth or development of a type of cancer in a human individual, comprising administering to a human individual who does not have cancer a first composition comprising an amount of a purified first complex of less than 50 micrograms effective to inhibit growth or development of said type of cancer, said first complex consisting essentially of an hsp90 noncovalently bound to a first antigenic molecule, in which either (a) the first complex is obtained from cancerous tissue of said type or metastasis thereof, or (b) the first antigenic molecule displays antigenicity of a tumor-specific antigen of said type of cancer.
 2. The method according to claim 1, in which the amount of the first complex is in the range of 5 to 49 micrograms.
 3. The method according to claim 1, in which the amount of the first complex is in the range of 5 to 40 micrograms.
 4. The method according to claim 1 in which the first antigenic molecule is a peptide with which the hsp is endogenously associated in vivo.
 5. The method according to claim 4 in which the first complex is prepared from said type of cancerous tissue or a metastasis thereof.
 6. The method according to claim 1 in which the first complex is produced in vitro.
 7. The method according to claim 6 in which the first antigenic molecule is a tumor-specific antigen of said type of cancer.
 8. The method according to claim 1 which further comprises administering to the individual a second composition comprising antigen presenting cells sensitized in vitro with a sensitizing amount of a second complex of a second hsp noncovalently bound to a second antigenic molecule, in which either (a) the second complex is obtained from cancerous tissue of said type or a metastasis thereof, or (b) the second antigenic molecule is a tumor-specific antigen of said type of cancer, and in which said sensitized antigen presenting cells are administered before, concurrently or after administration of the first complex.
 9. The method according to claim 8 in which said second hsp is selected from the group consisting of hsp70, hsp90, gp96 and combinations of the foregoing.
 10. The method according to claim 8 in which the first and second complexes are the same.
 11. The method according to claim 8 in which the amount of the first complex is in the range of 5 to 49 micrograms.
 12. The method according to claim 8 in which the amount of the first complex is in the range of 5 to 40 micrograms.
 13. The method according to claim 8 in which said second antigenic molecule is a peptide with which said second heat shock protein is endogenously associated in vivo.
 14. The method according to claim 8 in which the antigen presenting cells comprise macrophages. 